Background. HLA-DQ mismatch has been identified as a predictor of de novo donor-specific HLA antibody formation and antibody-mediated rejection. There are insufficient data to guide the incorporation of DQ mismatch into organ allocation decisions. Methods. We used a retrospective longitudinal cohort of adult living donor kidney transplant recipients from 11 centers across the United States for whom high-resolution class II typing was available. HLA-DQαβ heterodimer allele mismatch was quantified for all donor-recipient pairs, and outcome data were obtained through linkage with the Scientific Registry of Transplant Recipients. Results. We studied 3916 donor-recipient pairs. Recipient characteristics were notable for a median age of 51 (38–61) y, primarily unsensitized, with 74.5% of the cohort having 0% calculated panel-reactive antibody, and 60.4% with private insurance, for a median follow-up time of 5.86 y. We found that the HLA-DQαβ allele and HLA-DR antigen mismatch were each individually associated with an increased hazard of all-cause graft failure (adjusted hazard ratio [aHR] DQ = 1.03 1.14 1.28; aHR DR = 1.03 1.15 1.328), death-censored graft failure (aHR DQ =1.01 1.19 1.40; aHR DR = 0.099 1.18 1.39), and rejection. Having 2 HLA-DQαβ allele mismatches further increased the hazard of rejection even when controlling for HLA-DR mismatch (aHR 1.03 1.68 2.74). Conclusions. HLA-DQαβ allele mismatch predicted allograft rejection even when controlling for HLA-DR antigen mismatch and were both independently associated with increased risk of graft failure or rejection in adult living kidney transplant recipients. Given the strong burden of disease arising from the HLA-DQ antibody formation, we suggest that HLA-DQαβ should be prioritized over HLA-DR in donor selection.

To demonstrate the effect of pregnancy on panel reactive antibody (PRA) levels in women with repaired congenital heart disease (CHD). This will assess the utility of PRA assessment in reproductive age women and impact decision making regarding pursuit of pregnancy. Prospective observational cohort study of PRA levels in adult women with repaired CHD who have had pregnancies compared to those without pregnancies. Cardiac history and exposures reviewed. Serum PRA level analysis performed using the Luminex platform. A threshold of 25 percent was used to define PRA sensitization. Comparison groups were similar with regard to age, height, weight, prior sexual encounters, number of prior cardiac surgeries and times on cardiopulmonary (CP) bypass. Fontan physiology was analyzed as a prespecified subgroup. There is no significant difference in the rate of PRA sensitization when comparing women with a history of a prior pregnancy to those without a prior pregnancy. No difference in PRA sensitization was found by number of prior parity events. Individual PRA subtypes (I and II) also demonstrate no difference. The subgroup analysis of patients with Fontan physiology showed similar rates of PRA sensitization regardless of pregnancy history. Age is correlated to PRA-2 level (r=0.42, p=0.03). In women with repaired CHD, there is no association between history of pregnancy and rate of PRA sensitization. This study was only powered to detect a 45% difference in the rate of PRA sensitization and cannot exclude a smaller effect size. Future larger prospective and longitudinal studies on the effect of pregnancy events on PRA sensitization on an individual level are important.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Testing for anti-human leukocyte antigen (HLA) antibodies has now become standard practice in allogeneic hematopoietic stem cell transplantation (HSCT), and anti-HLA antibodies (both donor specific and non-donor specific) are being identified and have many potential consequences. Most studies suggest that donor-specific HLA antibodies lead to adverse outcomes, though little is reported on non-donor specific anti-HLA antibodies. We present the results of a retrospective cohort analysis of 157 patients who received HSCT at the University of Rochester over a period of four years. We identified 45 patients (28.7%) who had detectable anti-HLA antibodies, while only one patient (0.6%) had donor-specific anti-HLA antibodies. Patients with prior pregnancies and multiple transfusions were at increased risk to develop antibodies. In our cohort, the presence of non-donor specific anti-HLA antibodies did not significantly impact overall survival, progression free survival, graft failure, or transplant-related mortality.

In the US, the incidence of burn injury is > 2 million cases/year. A significant amount of burn related morbidity and mortality is due in part to immune dysfunction and wound healing complications which leads to increased risks of infection, coagulopathy, multi-organ failure, and sepsis. We conducted a prospective observational study of burn patients (≥18y). Citrated samples were collected at patient presentation. Whole blood aggregation (WBAgg) studies were performed immediately. TEGs were ordered as clinically indicated. Additional samples were collected at days 3, 7, 10, 14, 21, and 28, processed immediately, and plasma was kept frozen at -80oC until analysis. Samples were studied for microRNA, cytokines/chemokines, coagulation factors, and microparticles (MPs) concentration and identity. 57 patients have been enrolled in this study. The majority of patients had burns caused by flame exposure. The mean (± SD) age of patients was 47±16 and 70% were male. The mean total body surface area (TBSA) burn was 9.7±9.5% (range 0.3-76.0%). Most patients had abnormal WBAgg in response to ADP, collagen, and arachidonic acid. This was very obvious even in patients with as little as 1% TBSA. Significant increase in platelet counts were detected over the 28 days follow up. Most TEG tracings were normal or had shortened R times indicating hypercoagulable state. Higher TBSA% were associated with increased MP concentrations. Decreased IL-1α and IL-7 and increased IL-10, MCP-1 and IL-1RA levels at presentation were also seen with higher TBSA% (p<0.05). IL-8 levels were increased for up to 14 days in higher TBSA%. Interestingly, xenografting was associated with decreased levels of IP-10, MDC, IL-10, and MCP-1. Lastly, subjects who developed infections were found to have higher levels of IL-1RA and IL-8 for up to 14 days (p<0.05). This is one of the first prospective burn studies evaluating coagulopathy and platelet function at time of presentation. The reason for decreased platelet activity in burn patients is unclear and further studies are ongoing. One theory is that the platelets may degranulate as a response to the acute phase reaction. This may produce higher percentage of platelet microparticles, which contribute to the hypercoagulable state that has been observed by others and by our TEG studies. Further analysis is undergoing to identifying MPs type. The levels of chemokines and cytokines changes is consistent with published data. Early xenografting was associated with reduced inflammation. Finally, levels of certain cytokines may predict or herald subsequent development of infection. To better understand the immune dysfunction that occurs in burn patients and to reduce the concomitant morbidity and mortality.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

Objectives To provide an overview of the complexities associated with the human leukocyte antigen (HLA)-mediated platelet refractoriness. HLA antibody detection technologies and limitations associated with methodologies are discussed. Methods A case scenario and review of relevant literature describing platelet refractoriness are presented, followed by a discussion of HLA antibody testing. Results Following diagnosis of HLA-mediated refractoriness, a decision is made regarding the approach to obtain the appropriate platelets. The panel reactive antibodies (PRA) % of the patient, HLA typing, and limitations of the HLA testing should be taken into account when deciding which type of product would be the best option for a given patient. Conclusions Following confirmation and review of HLA antibody testing, platelets are ordered based upon the PRA% and approach employed, HLA-matched platelets, antigen restricted platelets, or cross-matched platelets. The platelets are transfused and a posttransfusion increment count is monitored to determine transfusion success.

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Recently, the neddylation (NEDD8) and the mammalian target of rapamycin (mTOR) signaling pathways have emerged as promising pharmaceutical targets for AML therapy. However, the interaction of these two pathways remains unclear. Here we evaluated the effects of pevonedistat, an inhibitor of the NEDD8 activating enzyme (NAE), and sapanisertib (TAK-228), an inhibitor of mTORC1 and mTORC2 as single agents or in combination on AML cell lines. We found that inhibition of neddylation with pevonedistat partially inhibited mTOR signaling transduction and vice versa, inhibition of mTOR signaling with sapanisertib partially inhibited neddylation in AML cell lines. Pevonedistat alone was able to induce cytotoxicity in most AML cell lines as well as in primary AML, whereas sapanisertib alone decreased cell metabolic activity, reduced cell size and arrested cells in G0 phase with only minimal induction of cell death. In addition, pevonedistat was able to induce cell differentiation, arrest cells in G2/M cell cycle phases, and induce DNA re-replication and damage. However, co-treatment with sapanisertib suppressed pevonedistat induced apoptosis, differentiation, S/G2/M arrest, and DNA damage. Taken together, our data demonstrate that pevonedistat and sapanisertib exhibit distinct anti-tumor effects on AML cells, i.e. cytotoxic and cytostatic effects, respectively; however, sapanisertib can attenuate pevonedistat-induced cellular responses in AML cells. Understanding mTOR and neddylation pathway interaction could provide therapeutic strategies for treatment of AML and other malignancies.

Platelet transfusion refractoriness in patients ranges from 7 to 34% and the etiology is typically attributable to formation of HLA antibodies from pregnancy or previous transfusion. Antibody development in children <9 months old is extremely uncommon due to their immature immune system. We report platelet refractoriness in a 5 month old male with hypoplastic left heart. Status post the Glenn procedure, the patient developed pulmonary hypertension and difficulty with oxygenation and ventilation. Eventually, he required placement on extracorporeal membrane oxygenation (ECMO). During the Glenn and later chest closure, the patient required transfusion with 4 units of RBCs for bleeding. Placement on ECMO required priming with several units of RBCs and additional blood product transfusions. Several days after starting ECMO, the patient was noted to be increasingly thrombocytopenic requiring platelet transfusions. After surgery and up to this point, he had received 33 RBCs, 51 apheresis platelets, 10 plasma, and 2 packs of cryoprecipitate. The etiology of his platelet refractoriness was investigated. He was found to have numerous class 1 HLA antibodies using the Luminex Class 1 SAB with a cPRA of 92% (HLA A: 1, 3, 11, 23, 24, 29, 36, 66, and 80; B: 7, 8, 13, 27, 37, 41, 42, 44, 45, 47, 48, 54, 56, 59, 60, 61, 67, 72, 73, 76, 81, and 82). A complement-dependent cytotoxicity (CDC) assay was done and the cPRA was found to be 85%. A little over 1 week later, the class 1 SAB assay was repeated and the cPRA was found to be 63% (HLA A: 26, 32; B: 13, 35, 37, 46, 47, 49, 50, 51, 53, 55, 57, 58, 63, 64, 65, 71, 72, 75, and 78); however, the CDC assay cPRA was 93%. Most of the antibodies identified were different. During the interim period, the patient had received 11 RBCs, 13 platelets, and 1 pack of cryoprecipitate. Thus, given the immaturity of the child’s immune system, his severe illness, and the dissimilarity between the testing results, the HLA antibodies were believed to have been transfused with various blood products. Per AABB guidelines, all platelet donors are tested for HLA antibodies if they have ever been pregnant or transfused before. Unfortunately, this child passed away from cardiac complications prior to further evaluation. This case serves to highlight that infants and very young children with immature immune systems who have been transfused blood products can be platelet refractory due to acquired HLA antibodies.

Platelet transfusion refractoriness in patients with hematological malignancies ranges from 7% to 34%. The etiology of platelet refractoriness in this patient population is typically attributable to due to formation of HLA antibodies (Abs) from pregnancy or previous transfusion. We report platelet refractoriness in the setting of induction chemotherapy for AML from allo-Abs to NAKa antigen/CD36/glycoprotein (GP) IV. A 66yo AAF presented with transfusion dependent AML with 70% circulating peripheral blasts and started on 7 + 3 regimen. She developed profound thrombocytopenia and her platelet count was 0 by day 13. As part of her evaluation for refractoriness, HLA antibody screening was done, but was negative. An Immucor ELISA assay was then performed and revealed positive Abs to GPIV (NAKa antigen/CD36). Abs against GPIIb/IIIa, GPIa/IIa, GPIb/IX, and HLA Class I were all negative. GPIV Abs were confirmed by Blood Center of Wisconsin using monoclonal Ab MBC 131.7 against CD36. Cross-matching was performed at our institution with multiple single donor apheresis platelets, all showing strong positive reactivity. Because CD36 in these units may be soluble, an incompatible leukoreduced, irradiated and washed single donor platelet unit was given which was tolerated well with a resultant increase to 14,000/μL. Despite strong reactivity of this unit in the crossmatch, the patient's platelet count increased and 1 week later was 16,000/μL despite no transfusion support. Platelet refractoriness in the setting of hematological malignancy is well documented. Abs develop from prior pregnancy and transfusion in the setting of prolonged pancytopenia in hematological malignancies. Typically, allo-Abs develop against platelet membrane structures including human platelet antigens, HLA antigens, and rarely NAKa antigen on the CD36 glycoprotein molecule. CD36 is expressed in almost 100% of white Europeans and is not detectably expressed by 2% of sub-Saharan Africans and 10% of Asians. CD36 is a soluble antigen and is expressed on various cells such as erythroblasts and monocytes. We propose that washing platelets may be a useful strategy in treating platelet refractoriness in patients with abs to NAKa antigen as finding CD36 negative donors is extremely difficult and few blood banks in North America test donors for this antigen.

To study the short and long term effects of transplanting hearts over positive B-cell flow crossmatches to patients with positive DSAs that become negative when DTT or molecular weight spin columns are used. Recipient and donor HLA typing was performed by rSSO-Luminex. Anti-HLA class-I and II antibodies by Single Antigen ((SAB) LABScreen Single Antigen Class-I and II) with and without molecular weight spin columns (MWSCs) or DTT. For identification of complement-fixing anti-HLA antibodies, the C1q test was used. Donor crossmatching was performed within 24 h of transplantation by flow cytometry (FXM) using serum drawn immediately before transplant. Patient allograft heart biopsy results and patient health were followed. Nine heart allograft recipients between 2012 and 2017 were identified that had either positive FXM and/or positive SAB I/II results that became negative when either DTT/MWSC were used (Table 1). In most, at least one of the antibodies identified was DSA. In 8, the DSA was also known to be a cryptic antigen. Some of the MFI values for DSA were below our cut-off of 3000 MFI. Patients were followed for rejection. Only one patient died from unrelated causes and never had any signs or biopsy results of cellular or antibody mediated rejection (AMR). The remaining patients are all alive and doing well. No patients ever had any biopsied signs of AMR. Several did have some cellular rejection that resolved with treatment however, rejection was not higher than in other heart transplant patients. This phenomenon was also observed in kidney transplantation. The use of MWSCs to evaluate positive FXM, SAB1, SAB2, and DSA in heart transplant candidates is effective in eliminating interference and cryptic antigens as well as determining which antibodies are "real" and pose a risk to the recipient. Here we show that use of MWSCs allows clarification of test results and gives one additional data on which to decide to transplant across DSA. Notably, all patients did well following transplant with no AMR.Download : Download high-res image (411KB)Download : Download full-size image

Our lab routinely treats high PRA patient serum with molecular weight centrifugal columns (SC) to remove high molecular weight interference from testing, especially in cases where prozone effect is suspected. Treated serum is used in Luminex single antigen bead (SAB) testing and/or flow cytometric crossmatching and results are correlated with test results using untreated sera. In this study our goal is to define the limitations and significance of sera treated by molecular weight centrifugal column. 124 patients had complement dependent cytotoxicity (CDC) and SAB/SC testing performed between January 2015 and February 2017. Protocol treats 250 μl of serum using the PALL 300 K Nanosep Centrifugal Device. Samples were centrifuged at 14,000 g for 15 min and collected from the reservoir for testing. Specificities determined using One Lambda LABScreen single antigen bead (SAB) Luminex assay and the GEN TRAK 60 well cell tray (CDC: 3 W plus AHG). Analysis included correlation of SAB/SC and CDC including evaluation of regraft status and donor specific antibody (DSA). 100/124 patients were CDC positive. All CDC specificities were also identified by SAB/SC testing. 46 were regrafts, and 38 (83%) had DSA identified by both CDC and SAB/SC. 9 of these regraft patients had the prozone effect eliminated in SAB/SC testing with CDC specificities matching. 24/124 patients were CDC negative. Eight of these were regrafts, four had DSA identified by SAB/SC, three were also negative by SAB (SC); all had some reactivity on SAB, but it was weak or against putative cryptic antigens. Our initial idea for use for the 300 K Nanosep centrifugal column (SC) was to treat sera to remove interference of high molecular weight substances including the IgM pentamers and eliminate any potential prozone effect. The concern is that IgG specific HLA antibody might also be lost in the process. Results confirm that all allo HLA antibody and DSA identified by CDC were preserved and positively identified by SAB/SC. 9 patients had prozone effect alleviated with SC treatment. One regraft patient had flow crossmatch results using SC sera that confirmed DSA remained positive. SAB/SC results MFI do not correlate to standard SAB results MFI. Interpretation should include pattern and history.