Donor WBCs Research Articles

Magnetism-Induced Cell-Targeting Transplantation Improves Early Hematopoietic Reconstitution in a Murine Bone Marrow Transplantation Model

BackgroundEarly hematopoietic reconstitution is essential for improving survival and reducing complications after hematopoietic stem/progenitor cell (HSC/HPC) transplantation (HSCT/HPCT). Increasing HSC/HPC homing to the bone marrow is a potential approach for promoting hematopoietic reconstitution.MethodsWe proposed the transplantation of HSCs/HPCs with a magnetism-induced cell-targeting transplantation (MagIC-TT) strategy. HSCs/HPCs were magnetized with CD45 microbeads. The biological characteristics (morphology, proliferation, viability, and ferroptosis) and target migration ability of these cells were studied in vitro. The hematopoietic reconstitution experiments were constructed in vivo in autologous and allogeneic bone marrow transplantation models with grouping showed as Table 1. The therapeutic effects were assessed by survival, donor chimerism, routine blood examination and histological analysis. We also performed transcriptomic sequencing for further mechanistic studies.ResultsThe biological characteristics was found no significant difference between the MagIC-TT and non-MagIC-TT groups, while migration ability was greatly improved with MagIC-TT (Data not showed). The survival rate was higher in the MagIC-TT group and significantly different in the allogeneic model (P<0.05). Hematopoietic reconstitution of donor chimerism, WBCs, HGB, and PLTs was faster in the MagIC-TT groups (within 22 days) (Figure 1). Confocal observation showed that more donor cells (eGFP +) were retained in the injected femur of the MagIC-TT group than in the femur of the non-MagIC-TT group 7 days after transplantation (P<0.05) (Figure 2). The severity of aGVHD (Assessed by survival, body weight, back arching, fur losting, diarrhea etc.) was reduced in the MagIC-TT groups in the allogeneic model (P<0.05) (Data not showed). Transcriptome sequencing revealed differentially expressed genes (DEGs) involved in localization/locomotion and pathways, cytokine-cytokine receptor interactions and chemokine signaling pathways between the two groups (Figure 3).ConclusionThe MagIC-TT strategy improves HSC/HPC homing, resulting in faster hematopoietic reconstitution in a murine bone marrow transplantation model.Key wordsMagnetism; Hematopoietic Stem Cell Transplantation (HSCT); Cell Homing; Hematopoietic Reconstitution; Ferroptosis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Total platelet donation count and donation frequency are determinants of plateletpheresis-associated lymphopenia.

Plateletpheresis using a leukocyte reduction system (LRS) traps donor WBCs in the LRS chamber, which may lead to lymphopenia, especially in frequent plateletpheresis donors. It seems plausible that this might cause adverse effects. However, current knowledge about potential confounders and donor health impacts is incomplete. Recent platelet donors and donations collected at University Hospital Regensburg from 2016 to 2019 using the Terumo BCT Trima Accel LRS system were retrospectively analyzed and compared with historical platelet donors and donations collected mainly with Fresenius Kabi Amicus non-LRS system from 2010 to 2013. Additionally, recent donors were prospectively surveyed using a health-related topics questionnaire. Analysis of 819 recent donors with 11,254 blood counts and 1464 questionnaires and 1011 historical donors with 12,848 blood counts revealed that increased annual platelet donation frequencies were associated with decreased lymphocyte counts in both groups. Median lymphocyte counts in recent donors with no versus ≥24 previous annual donations declined from 2.0 to 1.2 × 103 /μL (p < 2.2 × 10-16 ), and those in historical donors with no versus ≥24 previous annual donations decreased from 2.0 to 1.5 × 103 /μL (p=6 × 10-4 ), respectively. The questionnaire results showed that donation frequency and lymphopenia were not associated with upper respiratory tract infection (URTI) incidence or duration, but platelet donors who concomitantly donated granulocytes had significantly shorter URTI durations than those who did not (p=.008). This study confirmed that plateletpheresis-associated lymphopenia occurs in LRS and to a lesser degree in non-LRS platelet donors, but revealed no evidence of a negative impact on donor health.

Abstract 5536: Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients

Abstract Background: Expression of PD-L1 on tumor and immune markers in tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, each alone has limited predictive utility. Multimodal characterization of both the tumor and host immune system is an unmet medical need for the improved prediction of response to immunotherapy. Metastatic lesions are likely to be undersampled and require a liquid biopsy, given tumor heterogeneity and evolution and temporal changes in the host immune system. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from lung cancer patients were collected and shipped to Epic Sciences. Contrived samples were also developed, using cancer cell lines spiked into healthy donor (HD) blood. Red blood cells were lysed and nucleated cells were plated onto glass slides. Slides were stained with DAPI and immune cocktails, then imaged. Targets included pan-CK, CD45, PD-L1, CD4, CD8, Ki-67, and TIM-3. Approximately 3 million nucleated cells per slide were examined through advanced digital pathology pipelines to detect and quantify changes in T-cell populations and to assess circulating tumor burden. Results: Epic Sciences' rare cell detection platform has an analytically validated limit of detection of 2 cells/mL of blood. Immuno-panels were developed to profile leukocyte subpopulations and CTCs from a single blood sample and feasibility was demonstrated in cell lines, HD and lung disease patient blood. Quantitation experiments showed that immunomagnetically purified CD8+ and CD4+ cells spiked in 3:1, 1:1, and 1:3 ratios were detected as 3.1:1, 1:1.1, and 1:3.1. Purified CD4 cells were also spiked into healthy donor WBCs at target ratios of 1-10%. Percentages of detected spike-in CD4 cells were linearly correlated with the target ratios with correlation coefficient (r) = 0.96. 22.8% (18/79) lung cancer patients had PD-L1+ CTCs detected, and the majority (91%) of patients had PD-L1+ leukocyte populations. Conclusions: The low limit of detection of the Epic Sciences CTC platform coupled with the ability to archive patient blood samples allowed for retrospective, precise quantification of leukocyte subpopulations and PD-L1 expression on CTCs. Development of a liquid biopsy-based platform that can simultaneously measure immune biomarkers in CTCs as well as on leukocytes will allow for real-time assessment and monitoring of response to immune checkpoint inhibitors. Citation Format: Rachel Krupa, David Lu, Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Robin Richardson, Sean Nisperos, Ryon Graf, Jiyun Byun, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5536.

Microchimerism: a lasting legacy of transfusion?

Microchimerism: a lasting legacy of transfusion?

Supernatant from stored red blood cell primes inflammatory cells: influence of prestorage white cell reduction.

The contribution of RBC transfusion to adverse patient outcomes is controversial. There is conflicting clinical data and limited biologic data that provide an underpinning biologic rationale for any adverse impacts from RBC transfusion. This study used in-vitro measures of PMN stimulation to determine the ability of supernatant from RBCs to stimulate allogeneic WBCs and to determine the influence of residual donor WBCs and storage time on the proinflammatory potential of RBCs. Three types of RBCs were assessed: standard non-WBC-reduced RBCs (S-RBCs), buffy coat-poor RBCs (BCP-RBCs), and prestorage WBC-filtered RBC (LF-RBCs). Supernatant was collected weekly up to Day 42 of storage. PMN priming by supernatant from RBCs was determined by three methods: induction of CD11b expression on PMNs, induction of IL-8 release from PMNs, and the chemotactic effect of supernatant on PMNs. Supernatant from S-RBCs induced the expression of CD11b on PMNs, primed PMNs to release IL-8, and was chemotactic for PMNs. The magnitude of this PMN-priming progressively amplified with storage time. In contrast, supernatant from BCP-RBCs or LF-RBCs did not significantly prime PMNs. The PMN-priming effect of supernatant from RBCs correlated more closely with the level of MNCs in the RBCs than PMN content. Supernatant from stored S-RBCs prime unstimulated allogeneic PMNs in vitro. Prestorage buffy-coat WBC reduction was as effective as WBC depletion in abrogating this proinflammatory response elicited by supernatants from RBCs. The clinical consequences, if any, of these findings for transfusion recipients are unknown.

The effect of exposing murine splenocytes to UVB light, psoralen plus UVA light, or gamma-irradiation on in vitro and in vivo immune responses.

WBCs in blood components are a major factor contributing to immune responses such as GVHD and alloimmunization that follow transfusion. Irradiation of blood components has been used to regulate the contribution of donor WBCs to these immune responses. A systematic comparison of how the exposure of lymphoid cells to gamma-irradiation, UVB light, or psoralen + UVA light (PUVA) effects immune response was conducted to better define the best type of irradiation to be used in different clinical settings. Murine spleen cells were irradiated with varying doses and tested for their in vitro ability to be activated, to proliferate in response to mitogen or allogeneic stimulator cells, or to serve as stimulator cells. Irradiated donor cells were also tested for in vivo generation of GVHD, induction of alloantibody production, induction of restricted cytolytic T lymphocytes, and persistence of irradiated cells. In general, increasing amounts of irradiation resulted in increased inhibition of in vitro and in vivo responses, although the doses required for inhibition differed from assay to assay. In contrast, irradiation of donor cells did not always result in inhibition of recipient alloantibody responses but was dependent on the donor-recipient combination that was studied. Control of the in vivo outcomes of transfusing allogeneic cells is regulated by the type and dose of irradiation used and by the genotype of the donor and/or recipient.

Donor WBCs can persist and transiently mediate immunologic function in a murine transfusion model: effects of irradiation, storage, and histocompatibility.

Donor WBCs are responsible for numerous transfusion complications, but little is known concerning the natural history of their clearance following transfusion or of their function in the recipient's circulation. A murine transfusion model was developed to investigate the effects of blood component characteristics and histocompatibility on donor WBC survival kinetics and function. To investigate the effects of storage and irradiation, fresh whole blood and blood stored for 1, 2, and 6 weeks at 4 degrees C, all from male C57b (H2K(b)) mice, was transfused to female Balb/c (H2K(d)) mice. To study the effect of histocompatibility, blood was also transfused from C57b mice to Balb/c, FVB, C3H, and SW (outbred) mice. To investigate the xenogeneic setting, blood from humans, rats, and rabbits was transfused to Balb/c mice. Samples were collected weekly after transfusion, and the donor WBCs were analyzed, targeting the Y-chromosome with quantitative PCR. To investigate donor WBC function, dinitrochlorobenzene (DNCB) sensitivity was induced in donor and recipient mice, and the transfusion recipients were observed for hypersensitivity to DNCB. Donor WBCs had reduced in vivo survival equivalent to their period of storage ex vivo at 4 degrees C. Irradiation of donor blood produced no observable difference in donor WBC survival. Allogeneic male donor WBCs persisted (100-<1 cell/microL) in female Balb/c recipient mice blood over 6 weeks. Donor WBC survival kinetics displayed an early MHC-dependent phase, which was followed by a more rapid phase that was not influenced by donor-recipient MHC differences. All donor WBCs were cleared within 24 to 48 hours. DNCB sensitivity was passed through transfusion, where it was transiently expressed in naive recipients. The clearance of donor WBCs in the murine transfusion model is much slower than that in humans. Allogeneic donor WBC clearance may be biphasic, involving MHC-dependent as well as MHC-independent mechanisms. DNCB sensitivity can be transferred transiently to a naive recipient.

A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry.

A multicenter study was conducted to evaluate the performance characteristics of flow cytometry and microfluorimetry for counting low concentrations of WBCs and to compare the results with Nageotte hemocytometry. A two-phase study involving 10 centers located in the United States and in Europe was performed. Coded samples of RBCs and platelets were distributed by 24-hour (Phase 1) or 2-day (Phase 2) courier service to each test site for analysis. Samples were prepared to include concentrations of WBCs slightly above and below the concentration corresponding to the threshold standards for WBC-reduced RBCs and platelets. All centers tested samples by Nageotte hemocytometry plus one or both of two automated methods. Both flow cytometry and microfluorometry gave better results than Nageotte hemocytometry in testing freshly prepared samples. At WBC concentrations >5 per microL (RBCs) or >3 per microL (platelets), the intersite CV was <20 percent for the automated methods but >30 percent for the Nageotte hemocytometer method (p<0.001). Accuracy was greater for the automated methods than for the Nageotte hemocytometer method (p<0. 001). Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with the automated methods. All methods had poorer performance in testing samples that required > or =2 days' shipment than in testing of those requiring overnight shipment. Automated methods for counting residual donor WBCs in WBC-reduced cellular components offer advantages of improved precision and greater accuracy than are seen with the Nageotte hemocytometer method. Automated methods are less labor-intensive but more costly than microscopic methods. Preparation and shipping methods will need further refinement for samples to be counted more than 24 hours after sample collection.

Survival of Donor Leukocyte Subpopulations in Immunocompetent Transfusion Recipients: Frequent Long-Term Microchimerism in Severe Trauma Patients

We recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4(+) male donor cells peaked at day 3 or 5 (0.01 to 1 cell/microL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/microL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.

Survival of Donor Leukocyte Subpopulations in Immunocompetent Transfusion Recipients: Frequent Long-Term Microchimerism in Severe Trauma Patients

AbstractWe recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4+ male donor cells peaked at day 3 or 5 (0.01 to 1 cell/μL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/μL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.

Unusual kinetics of white cell clearance in transfused mice

Donor white cells (WBCs) in blood transfusions are responsible for complications in recipients, including alloimmunization, graft-versus-host disease (GVHD), and virus transmission and reactivation. The recent use of sequence-specific polymerase chain reaction assays to monitor the kinetics of clearance of donor WBCs in transfused humans and dogs found transient recirculation of donor lymphocytes on Days 3 to 5 after transfusion; this presumably reflected an abortive GVHD reaction to major histocompatibility complex-incompatible recipient cells, after which donor WBCs were cleared to undetectable levels. This study sought to develop a murine model to further characterize the kinetics and major histocompatibility complex restriction of donor WBC clearance. A sensitive murine Y chromosome-specific polymerase chain reaction assay was developed and applied to serial blood samples collected after transfusions of allogeneic blood to naive inbred, primed inbred, and outbred mice, as well as after transfusions of gamma-radiated blood to naive inbred mice. In inbred mice, both naive and primed to the allogeneic blood donor, transfused WBCs were not cleared to undetectable levels for more than 1 month after transfusion. Transfused outbred mice also showed prolonged donor WBC survival, although at lower levels than inbred mice. There was no evidence of GVHD in either inbred or outbred mice, and gamma radiation had no significant impact on donor WBC persistence. These results contrast with the rapid clearance of donor WBCs observed in humans and dogs. The immunologic basis for this discrepancy remains unclear. Caution should be exercised in any extrapolation to humans of conclusions drawn from results in transfused mice.

Low retention of white cell fragments by polyester fiber white cell-reduction platelet filters.

There is intense interest in the potential of current white cell (WBC)-reduction filters to prevent the alloimmunization of patients by the residual donor WBCs in filtered blood components transfused to them. Little attention has been paid to the capacity of current synthetic fiber filters to remove WBC membrane fragments bearing detectable leukocyte antigens. Fluorescein isothiocyanate-conjugated monoclonal antibody to a universal WBC membrane antigen (CD45) and high-speed centrifugation coupled with ficoll-hypaque differential sedimentation were used to quantitate low-density WBC fragments in single-donor platelet components before and after filtration to determine if a polyester fiber filter retained WBC fragments. Prefiltration measurements in 25 single-donor platelet components indicated that WBC fragments increased with length of storage up to 5 days at room temperature (p < 0.0001). When fragments in eight components were measured before and after filtration, absolute values for differences were insignificant (p = 0.15). WBC fragments were poorly retained by these polyester fiber WBC-reduction filters. The antigenicity of WBC fragments could contribute to the WBC alloimmunization of some recipients of WBC-reduced blood components.

Monocyte antigens may cause graft rejection

Monocyte cross matching should be done before patients undergo renal transplantation, according to James Cerilli, MD, professor of surgery at Ohio State University. The technique involves obtaining an almost pure monocyte preparation from the kidney donor's WBCs and mixing this preparation with the recipient's serum. Then, Cerilli says, You look to see if there is anything in the serum kills those cells. This technique, he adds, is only moderately more difficult than standard cross matching. During the annual meeting of the American Society of Transplant Surgeons in Chicago, Cerilli reported on a preliminary study designed to evaluate the relationship between renal allograft survival and results of monocyte cross matching. We attempted to show, he explains, that patients who have antibody to [donor] monocytes do poorly, even when all the other immunologic parameters predict an excellent outcome. Earlier investigations by Peter Stastny, MD, of the University of Texas at Dallas