Detection Of Chimerism Research Articles

Symptotic detection of chimerism

Microchimerism (MC), transplacental acquisition of allogeneic cells from the mother (maternofetal MC) or from the fetus (fetomaternal MC) has been in the focus of research recently. Amplicons using Y-chromosome specific SRY and DYS14 sequences have been used as markers to trace cells from a male fetus in the mother. The sensitivity of these markers in formaldehyde fixed paraffin embedded samples, however, is less than optimal. To study chimerism in breast cancer we took advantage of the evolutionary history of the Y chromosome and designed amplicons on gene repeats to generate additive PCR signals. The increased sensitivity detected high incidence of male chimerism in normal breast tissues. We also showed correlation with protection from cancer with unique quantitative biology. Accumulating data from biology and medicine indicate that natural chimerism is astonishingly frequent and may affect human conditions. We hypothesize that it has significant evolutionary ramifications as well.

High resolution melting analysis of deletion/insertion polymorphisms: A new method for the detection and quantification of mixed chimerism in allogeneic stem cell transplantation

Increasing mixed chimerism after allogeneic stem cell transplantation has been associated with a high risk of relapse and probable graft failure in patient with hematological malignancies as well as non-malignant conditions. We evaluated a new method for chimerism detection, based on the quantitative High Resolution Melting Analysis (HRMA) of deletion/insertion polymorphisms (DIPs).The study consisted in the selection of a panel of DIPs, all generating genotype-specific melting curves, and in the use of samples containing opposite molecular species (homozygous INS/INS and DEL/DEL) mixed in different percentages to create a standard curve for each polymorphism.The detection of mixed chimerism with the HRMA attained a sensitivity of <1%, as well as good accuracy and precision with Percent Errors and Coefficients of Variation not exceeding 30% in reconstruction experiments with DNA mixtures.The present approach provides accurate and precise estimates of mixed chimerism and makes the method open to evaluation for its use in clinical practice.

A fast and simple approach for the simultaneous detection of hematopoietic chimerism, NPM1, and FLT3-ITD mutations after allogeneic stem cell transplantation

Hematopoietic chimerism can be used as a tool for patient management after allogeneic hematopoietic stem cell transplantation (HSCT). An increase in the proportion of recipient cells after transplantation is strongly associated with relapse in chronic myeloid leukemia. However, in acute myeloid leukemia (AML) the significance of increasing mixed chimerism (MC) as a predictive marker for relapse is less clear. Several mutations frequently found in AML have been employed for minimal residual disease detection and relapse prediction. Therefore, a combined analysis of hematopoietic chimerism and of the molecular aberrations found in AML could be used to improve MC characterization. We developed a multiplex PCR for use in the simultaneous detection of hematopoietic chimerism and mutations in nucleophosmin (NPM1) and fms-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD). A total of 303 samples from 20 AML patients were analyzed after HSCT. The microsatellite markers used for hematopoietic chimerism detection were D1S80, D7S1517, D4S2366, THO1, and SE33. A total of 149 samples from 18 patients showed MC with a mean detection time of 9.7 months. From the 18 patients with MC, in 6 of the patients, no FLT3-ITD or NPM1 mutation was found at any time point tested, and these patients remained in complete hematological remission. In 12 patients with MC, FLT3-ITD and NPM1 mutations were found, and these patients showed signs of hematological relapse. Our combined analysis of NPM1/FLT3-ITD mutations and hematopoietic chimerism improved the characterization of patients with MC after HSCT. The present approach may be further expanded by combining additional mutations found in AML with hematopoietic chimerism detection.

Detection and quantification of chimerism by droplet digital PCR

Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis.

Diagnostic and Prognostic Value of Mitochondrial DNA Minisatellites after Stem Cell Transplantation

Mitochondrial DNA has been used to investigate phylogenetic relationships and pathophysiologic roles in aging, degenerative diseases, and cancer. We investigated the prognostic usefulness of mitochondrial DNA minisatellite (mtMS) markers compared with nuclear short tandem repeat markers by evaluating the laboratory performance and clinical value of these markers in a large sample of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) with various simulated conditions in vitro and in serial follow-up samples. We examined the value of mtMS markers as a prognostic indicator in 100 patients with various hematologic disorders undergoing allo-HSCT, including 35 patients with longitudinal follow-up for 55 months. The mtMS markers showed high sensitivity and accuracy for the quantitative detection of chimerism compared with nuclear short tandem repeat markers, particularly in unrelated transplantation and under inappropriate sampling conditions. Longitudinal follow-up after allo-HSCT disclosed that chimerism precisely reflected the status of engraftment or relapse during the clinicopathological course. Moreover, changes in mtMS markers in recipients before allo-HSCT were associated with clinical outcomes. Our data indicate that mtMS markers have multiple functions in monitoring mixed chimerism and predicting prognosis after allo-HSCT.

Comparison of different blood sample processing methods for sensitive detection of low level chimerism byRHDreal-time PCR assay

The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.

Follow-up of the patients after bone marrow transplantation for red blood cell chimerism using flow cytometry

The importance of chimerism is that it is useful to demonstrate engraftment. It allows for early intervention which might facilitate treatment of emerging relapse in leukemia, and it may help to prevent graft rejection. The aim of this study is to evaluate mixed red cell population and red blood cell chimerism after hematopoietic stem cell transplantation to predict the final outcome of mixed chimerism which may help in deciding interventions and preventing graft rejection. This study was conducted on 22 bone marrow-transplanted patients admitted to Bone Marrow Transplantation (BMT) Unit, Nasser institute, using simple agglutination method and flow cytometery (FCM) technique. In our study, three recipients had complete chimerism after 6 month which was detected by agglutination method, and one recipient had complete chimerism after 1 year which was detected by both simple agglutination method and FCM. Two recipients had mixed-field agglutination after 30 days which was detected by agglutination method and FCM. Evaluating erythrocyte repopulation by the agglutination method is feasible, easy, and cost-effective. FCM analysis is a simple, sensitive test and an objective method, which could be used as a protocol for follow-up to detect chimerism after allogeneic BMT. However, due to the difficulty found in retrieving the patients for follow-up, we suggest the use of alternative methods for detection of chimerism as variable number of tandem repeats and XY fluorescence in situ hybridization probe.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Chimerism detection by short tandem repeat analysis when donor and recipient genotypes are not known

BackgroundsThe analysis of chimerism after bone marrow transplantation by STR–PCR is frequently carried out with commercial kits designed for forensic purposes and including too many non informative STR. Furthermore, in routine clinical practice it is not uncommon to lack the pre-transplant genotype of the recipient or the donor, thus making it difficult to identify both components in the post-transplant genotype. The objective of this paper is to overcome these drawbacks by analyzing the informativity of STR markers from a perspective which can be applied whether the pretransplant genotypes are available or not, and selecting a minimum STR panel that allows an effective direct detection of chimerism. MethodsDNA extraction, STR–PCR and fragment analysis of 15 STR in 90 donor-recipient pairs, 60 of which were part of the discovery set and 30 in a validation set. Loci were considered as informative when there were 3 or 4 different alleles in the combined genotypes of the recipient and the donor. ResultsThe informativity varied between 41.6 and 76.6. The 4 most informative loci were D2S1338, D21S11, D18S51 and FGA. We could select a minimum set of 8 markers (D2S1338, D21S11, D18S51, FGA, VWA, D19S433, TH01 and D3S1358) that provided at least 3 informative loci in 95% of cases. ConclusionThis minimum STR panel may be an efficient way to detect and quantitate donor–recipient chimerism after transplantation.

Monitoring the Chimerism Status of Subpopulation in HLA Haplo-Identical Stem Cell Transplantation Using the Anti HLA Antibodies: Based on the Study of HLA Distribution in Korean Population

Abstract 4706 Background:Determining the chimerism in stem cell transplantation (SCT) is important in the monitoring of engraftment. Conventional monitoring methods such as short tandem repeat (STR) PCR are labor intensive and difficult in showing the changes of cell subpopulations. Nowadays, HLA-haploidentical SCT is performed actively and flow cytometric analysis using anti HLA antibody targeted for the different HLA between donor and recipient in HLA-haploidentical SCT can be useful by observing changes of cell subpopulations and determining the chimerism. Methods:Based on the study of HLA distribution in Korean population, we designed the panel of HLA monoclonal antibody that can detect and differentiate major HLA of Korean population. This panel was verified against 23 cases of HLA-haploidentical SCT. Flow cytometric analysis was performed using anti-HLA-antibodies on one pediatric patient with aplastic anemia who underwent HLA-haploidentical SCT. Tests were performed every day since SCT. The appearance or disappearance of donor and/or recipient HLA was observed using antibodies against mismatched HLA. Donor and/or recipient T cells, B cells, NK cells, and monocytes were observed using CD3, CD19, CD56, and CD13 respectively. The flow cytometric results were interpreted by observing the changes in subpopulations of detected cells and determining the engraftment status of patients. Results:A total of 11 anti-HLA-A-antibodies and anti-HLA-B-antibodies were selected. They cover 97.9% of HLA-A and 8.6% of HLA-B in Korean population. In the verification of our panel of HLA monoclonal antibody, we distinguished donor and recipient cells in 21 of 23 HLA-haploidentical SCT cases. In our case, the patient had HLA-A2/A24 while the donor had HLA-A2/A33. Recipient type of CD3(+) and HLA-A33(-) T cells appeared in first few days but donor type CD3(+) and HLA-A33(+) T cells appeared at day 20 and were increased. Donor type CD56(+) and HLA-A33(+) cells were seen at analysis done in day 8 and continued to appear. HLA-A33(+) cells were consistently observed and increased throughout the follow up period, showing the process of engraftment of donor stem cells. Conclusion:We were able to apply our flow cytometric analysis design using HLA monoclonal antibody for the detection of chimerism in HLA-haploidentical SCT. This method was more simple and sensitive than conventional monitoring techniques. Moreover, this method allowed us to observe the dynamics of changes in cell subpopulations after HLA-haploidentical SCT. Flow cytometric analysis can be considered as a strong tool for observing the changes in cell subpopulations and the monitoring the engraftment of HLA-haploidentical SCT. Disclosures:No relevant conflicts of interest to declare.

Fetal Human Liver Progenitor Cells: A Potential Immune-Privileged Cellular Source from Non Heart Beating Donors

Fetal Human Liver Progenitor Cells: A Potential Immune-Privileged Cellular Source from Non Heart Beating Donors

Correlation of Chimerism with Acute Graft-versus-Host Disease in Rats following Liver Transplantation

The accurate diagnosis of acute graft-versus-host disease following liver transplantation (LTx-aGVHD) has been hampered. Chimerism appears in the majority of recipients after LT and its significance in the diagnosis of LTx-aGVHD has not been clearly established. To demonstrate the significance of chimerism on the diagnosis of LTx-aGVHD, we compared the change of chimerism in syngeneic LT recipients, semiallogeneic LT recipients, and LTx-aGVHD induced recipients. Chimerism in PBMCs following sex-mismatched LT was identified by real-time PCR based on a rat Y-chromosome-specific primer. All recipients in semiallogeneic group grew in a normal pattern. However, when 4 × 108 donor splenocytes were transferred simultaneously during LT, the morbidity of lethal aGVHD was 100%. The chimerism appeared slightly higher in the semiallogeneic group than in the syngeneic LT group, but the difference was not significant. However, when the recipients developed lethal aGVHD after LT, chimerism in the PBMCs increased progressively, and even at an early time, a significant increase in chimerism was observed. In conclusion, high level chimerism correlated well with LTx-aGVHD, and detection of chimerism soon after transplantation may be of value in the diagnosis of LTx-aGVHD prior to the onset of symptoms.

Minimal Residual Disease Diagnostics and Chimerism in the Post-Transplant Period in Acute Myeloid Leukemia

In acute myeloid leukemia (AML), the selection of poor-risk patients for allogeneic hematopoietic stem cell transplantation (HSCT) is associated with rather high post-transplant relapse rates. As immunotherapeutic intervention is considered to be more effective before the cytomorphologic manifestation of relapse, post-transplant monitoring gains increasing attention in stem cell recipients with a previous diagnosis of AML. Different methods for detection of chimerism (e.g., microsatellite analysis or quantitative real-time PCR) are available to quantify the ratio of donor and recipient cells in the post-transplant period. Various studies demonstrated the potential use of mixed chimerism kinetics to predict relapse of the AML. CD34+-specific chimerism is associated with a higher specificity of chimerism analysis. Nevertheless, a decrease of donor cells can have other causes as well. Therefore, efforts continue to introduce minimal residual disease (MRD) monitoring based on molecular mutations in the post-transplant period. The NPM1 (nucleophosmin) mutations can be monitored by sensitive quantitative real-time PCR in subsets of stem cell recipients with AML, but for approximately 20% of patients, suitable molecular mutations for post-transplant MRD monitoring are not available so far. This emphasizes the need for an expansion of the panel of MRD markers in the transplant setting.

Detection of human hematopoietic stem cell engraftment in the livers of adult immunodeficient mice by an optimized flow cytometric method.

Immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice are a valuable resource to study human hematopoietic stem cells. Prolonged multilineage hematopoiesis indicates stem cell engraftment and generally is measured by flow cytometry. In this study, we took advantage of the multi-parameter detection afforded by modern flow cytometers to optimize detection of human hematopoiesis in NSG mice. Antigens widely expressed by mouse or human cells were evaluated as markers to distinguish mixtures of these cells to optimize and test the limits of chimerism detection. The bone marrow, spleen, and liver of NSG mice transplanted with human hematopoietic cells were analyzed for evidence of engraftment.Mouse bone marrow cells were best marked for exclusion by staining with a combination of CD45, TER-119, and anti-H-2K(d) monoclonal antibodies, whereas live human cells were most accurately identified by elimination of cell doublets and positive staining for CD59. Human stem cells (CD34(++)CD133(+)CD38(low)) and progenitors were detected in the bone marrow and liver, but not in the spleen. An unusual pattern of myeloid antigen expression was detected in the bone marrow and CD3(+)CD4(+)CD8(+) T-cells were detected in the spleen. We concluded that multicolor flow cytometric analysis that clearly distinguishes mouse and human cells offers accurate detection of human chimerism in NSG mice. Human hematopoiesis can be detected in the bone marrow and liver of NSG mice with T-lymphopoiesis, possibly occurring in the spleen.

Suivi de chimérisme des populations cellulaires myéloïdes chez des patients allogreffés atteints de leucémie aiguë myéloïde : étude lyonnaise

Chimerism analysis after allogeneic haematopoietic stem cell transplantation has been used to document engraftment and to adapt therapy promptly. The aim of this study was to document engraftment and to detect as soon as possible relapse in patients with acute myeloid leukaemia who underwent stem cell transplantation. Real-time quantitative polymerase chain reaction is a highly sensitive and reproducible technology. It is useful in some disease to target selected sub-populations in order to have an earlier detection of relapse on cell fractions. In the acute myeloid leukaemia (n=65), analysis of the chimerism on whole peripheral blood cells and bone marrow cells, CD3+ cells, specific myeloid CD33+ cells (from blood) and CD34+ cells (from bone marrow) is of importance. After transplant, 25 patients relapsed (38%), three massively, with chimerism detection in whole blood and bone marrow and 22 insidiously following two different schemes (GRI and GRII). In GRI, (n=13): chimerism of CD33+ and CD34+ cellular fractions allowed an early detection of relapse in 100% of cases undetected in whole cells whereas in GRII (n=9): myeloid cells could identified relapse in 89% of cases when whole blood cells and CD3+ cells expressed a mixed chimerism. This study highlighted the importance of sub-cellular population chimerism documentation enable to ascertain a stable engraftment and to detect early relapse. The selection of sub-cellular population studied with high sensitive technology allows a rapid and efficient intervention before the onset of clinical signs in patient with acute myeloid leukaemia and could improve the patient's follow-up.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.

Effects of donor-derived spleen cells combined with anti CD154 monoclonal antibody on bone marrow transplantation in non-myeloablative conditioning regimen for the induction of allogeneic mixed chimerism

Objective To explore the role of donor spleen ceils(Sos)(including transfusion pathway)combined with anti CD154 monoclonal antibody(mAb)on bone marrow transplantation in nonmyeloablative pre-conditioning regimen for induction of aUngeneic mixed chimerism and donor-specific immune tolerance.Methods Lewis rats(Rt11)were used as donors,Wistar-Furth rats(Wistar,Rt1u)as recipients,and Sprague-Oawley(SD)rats as the third party donors.Scs and bone marrow cells(BMCs)were harvested from donor Lewis rats.Recipient Wistar rats were randomly divided into 4 groups,20 rats in each group.In group A,dorsum penis vein(i.v.)injection of Lewis rats 2×108 Scs on day 0 alone.In group B,portal vein(P.V.)injection of Lewis rats 2×108 Scs on day 0 alone.In group C,P.V.injection of Lewis rats 2×108 Scs on day 0,in combination with i.v.injection of Lewis rats 1×108 BMCs on day 3.In group D,P.V.injection of Lewis rats 2×108 Scs on day 0,intraperitoneal injection of 3 μg AH.F5 (hamster anti-rat CD154 mAb)2 h later,in combination with i.v.injection of Lewis rats 1×108 BMCs on day 3.Tolerance was assessed by stimulation index(SI)of one-way mixed lymphocyte reaction(MLR).Rt11 positive chimersim in recipient spleen and thymus was measured by flow cytometric(FACS)analysis on the day 20,40.In rats in group D were additionally subjected to chimerism detection on the day 100.Results SI in group D on the day 20,and 40 was significantly lower than other groups(4.43±0.19,4.97±0.25,P ＜0.01).The results of Rtl1+ assay revealed that there was no chimersim in group A.While chimersim was transient and lower in groups B and C on the day 20,and rapidly decreased.Only group D had stable and higher level chimerism in spleen(32.40±3.29)%,(23.06±3.77)% and thymus(31.08±2.60)%,(24.52±3.22)% on the day 20,and 40.Furthmore,long-term chimerism (＞ 100days)was observed in the group D.Conclusion High level of ehimerism(＞ 30% in peripheral spleen and ＞ 20% in central thymus)provides the foundation to BMCs to play a critical role in the induction or maintenance of tolerance.P.V.injection of Sos and anti CD154 mAb,plus i.v.injection of BMCs had synergistic effect to induce donor-specific tolerance and establish stable allogeneic mixed ehimersim,without participation of megadose BMCs,myleosuppressive drugs and irradiation. Key words: Bone marrow transplantation; Spleen cells; CD154; Chirnerism; Immune tolerance

Chimerism Detection In Hematopoietic Cell Transplant Recipients By Real-Time Quantitative Pcr (Q-PCR) Vs. Short Tandem Repeat (STR) Analysis

Chimerism Detection In Hematopoietic Cell Transplant Recipients By Real-Time Quantitative Pcr (Q-PCR) Vs. Short Tandem Repeat (STR) Analysis

IgG Allotypes Reveal That Antimicrobial Humoral Immunity Persists after Reduced-Intensity Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for hematologic malignancies through graft-versus-leukemia/lymphoma (GVL) responses and replacement of the patient's immune system. Conditioning intensity may influence hematopoietic reconstitution, persistent recipient immunity, and donor alloimmune responses. High-dose conditioning (HDC) causes rapid conversion to full donor T- and B-cell chimerism, while patients who undergo reduced-intensity conditioning (RIC) progress through a dynamic mixed chimerism extending weeks to months. We hypothesize that long-lived recipient plasma cells persist, providing beneficial antimicrobial serologic immunity, while donor B cells target allo-antigens, contributing to both GVL and GVHD. Furthermore, recipient humoral immunity persists longer following RIC than HDC, leading to fewer infections and decreased transplant-related mortality.Inherited polymorphisms in IgG heavy chain constant regions can be recognized by allotype-specific monoclonal antibodies and thus distinguish donor and recipient antibodies. We developed a quantitative ELISA by coating mouse monoclonal antibodies specific for the G1m(f) and G2m(n) allotypes. Serial dilutions of 63 patients and their donors were incubated and detected by alkaline phosphatase-conjugated anti-human IgG polyclonal antibody. Twenty-eight HDC patients were conditioned with VP-16 and total body irradiation, while 35 RIC patients received total lymphoid irradiation and anti-thymocyte globulin (TLI-ATG). One hundred fifteen of the total 126 subjects (91%) expressed G1m(f) (null allele frequency: 0.29), and 100 of 126 (79%) expressed G2m(n) (null allele frequency: 0.45). Twenty-six of the 63 pairs (41%) were informative because either the donor or recipient was homozygous null for an allotype. Following both RIC and HDC when the recipient was allotype null, the donor allotype was first detected six months post-HCT and reached 50–80% of donor levels by 12 months (n=8 pairs). Eighteen donors were homozygous allotype null, and recipient-specific antibody was prospectively determined. By 12 months after HDC, recipient antibody levels fell to <10% of pre-HCT allotype levels (n=11 pairs). In contrast, RIC patients retained the allotype at >90% of pre-HCT levels through 12 months post-HCT (n=7 pairs), demonstrating a significant difference from HDC patients in one-year median recipient allotype levels (p=0.016). We confirmed RIC humoral immune persistence by DNA chimerism detection of CD38+CD138+ plasma cells from bone marrow aspirates collected 12 months post-HCT. Analysis revealed 30–58% donor levels of plasma cells, suggesting that RIC patients maintain long-lived plasma cells.Allotype-informative patients were measured by ELISA for total IgG and allotype-specific IgG against Epstein-Barr virus (EBV), varicella zoster virus (VZV), pneumococcus, and influenza. HDC patients displayed loss of recipient antimicrobial allotype between five and eight months in conjunction with loss of bulk allotype. RIC patients maintained >90% pre-HCT allotype levels through one year correlating to levels of total IgG against all four infectious agents when they were seropositive before HCT. In addition, new donorspecific responses to influenza, pneumococcus, and VZV were detected in both sets of patients within seven to 14 months.In the allotype-informative setting, H-Y antibody analysis confirmed that donor-derived allogeneic antibodies against UTY2 and DDX3Y developed after nine and 12 months, respectively, and persisted in both HDC and RIC male patients with female donors.In summary, RIC recipients benefit from a twofold effect: persistent recipient-derived antimicrobial humoral immunity and donor-derived allogeneic B-cell responses. Our studies may explain why RIC results in decreased infectious complications in the post-HCT setting.

116-P: Chimerism detection by real-time PCR vs STR PCR

116-P: Chimerism detection by real-time PCR vs STR PCR