Number Of Total Nucleated Cells Research Articles

Particularities of the bone tissue regenerative properties of the proximal part of the tibia in patients with rheumatoid arthritis with multi-plane knee deformities

Background. Rheumatoid arthritis (RA) has no equal among the musculoskeletal system diseases in terms of the severity of joint damage. Knee joint damage was noted in 65–70 % of RA patients and it is the most common cause of disability. The purpose was to study the indices of osteogenic activity of stromal (mesenchymal) stem cells (MSCs) of the articular bone ends of the knee joint in patients with secondary knee osteoarthritis (KOA) due to RA in the presence of multi-plane deformations. Materials and methods. The studied material was samples of cancellous bone of the anterior and posterior parts of the plateau of the proximal part of the tibia and spongiosa from a depth of 1 cm and 3 cm from the articular surface, namely from the proximal epimetaphysis of the tibia, removed during knee arthroplasty of patients with secon­dary KOA due to RA and in patients with primary KOA in the presence of multi-plane deformations. Using the method of cloning colony-forming units of fibroblasts (CSF) of bone tissues, 38 patients were examined, including 9 patients with RA and 29 patients with primary KOA with damage to the knee joint in the presence of multi-plane deformations. Results. The total number of nucleated cells in 1 cm3 of spongiosa of the front part of the tibial plateau was 2.3 times lower compared to the parameter of this index in the posterior part (p < 0.05). The characteristics of spongy tissue in terms of the number of nucleated cells and the content of CSF in 1 cm3 from a depth of 3 cm from the articular surface of the proximal epimetaphysis of the tibia of patients with RA were almost 3 times higher compared to the indices of patients with primary KOA (p < 0.05). Conclusions. Changes in the renovation processes of bone tissue in patients with secondary KOA due to RA and in patients with primary KOA in the presence of multi-plane deformations are unidirectional, but their expressiveness is greater in patients with RA. At a depth of 3 cm from the articular surface of the proximal epimetaphysis of the tibia, the conditions for secondary fixation of the leg of the knee joint replacement are much better, all other conditions being equal, so the "survival" of the endoprosthesis with an elongated leg will be greater.

A prospective study of nanopore-targeted sequencing in the diagnosis of central nervous system infections.

This study aims to prospectively evaluate the ability of nanopore-targeted sequencing (NTS) in the detection of pathogens in cerebrospinal fluid (CSF). It was the first time combining mNGS and microbial culture to verify the NTS-positive results also using 16S rDNA amplification with Sanger sequencing. Although microbial culture was thought to be the gold standard for pathogens detection and diagnosis of infectious diseases, this study suggested that microbial culture of CSF is not the most appropriate way for diagnosing central nervous system (CNS) infection. NTS should be recommended to be used in CSF for diagnosing CNS infection. When evaluating the value of NTS for diagnosis of CNS infections, the results of CSF TNC should be combined, and NTS-positive result is observed to be more reliable in patients with CSF TNC level >10 cells/μL.

The Outcomes of Cord Blood Transplantations Infused with Higher Total Number of Nucleated Cells Were Equivalent to Unrelated Bone Marrow Transplantations

Introduction: Choosing a suitable donor is essential to achieve a good response for allogeneic hematopoietic cell transplantation (allo-HCT). Several previous studies have compared unrelated bone marrow transplantation (UR-BMT) and cord blood transplantation (CBT), but the results differed among each study, and their superiority has been controversial. The total number of nucleated cells (TNC) in cord blood unit is crucial for CBT to avoid early complications, including engraftment failure. The outcome of CBT may improve by choosing cord blood unit with a higher TNC. Methods: We retrospectively compared the outcomes of CBT with higher TNC (hCBT) and UR-BMT conducted in our institute from January 1st, 2013, to May 31st, 2023. Cut off point of TNC was the median value. The primary endpoint was overall survival (OS), and the secondary endpoints were non-relapse mortality (NRM), cumulative incidence of relapse, and cumulative incidence of complications. Results: We analyzed a total of 316 patients, including 139 patients who underwent CBT and 177 patients who received UR-BMT. The median infused TNC was 2.71 (range, 0.027-6.42) ×10^7/kg. hCBT group included 69 patients. Significantly more patients in the hCBT group had prior allo-HCTs (hCBT: 18.8%, UR-BMT: 5.6%, p=0.003), and hCBT procedures were more frequently conducted since 2018 (hCBT: 66.7%, UR-BMT: 36.7%, p<0.001). For each group, most patients received tacrolimus and short-term methotrexate as graft-versus-host disease prophylaxis (hCBT: 94.2%, UR-BMT: 95.5%, p=0.277). ABO matched donors were more commonly chosen for UR-BMT (hCBT: 24.6%, UR-BMT: 46.3%, p=0.006). Patients with myelodysplastic syndrome underwent UR-BMT more frequently (hCBT: 7.2%, UR-BMT: 20.9%). Patients with a high or very high disease risk index were more frequently included in the hCBT group (hCBT: 37.5%, UR-BMT: 27.9%, p=0.060). Patient sex, age, days from diagnosis to transplantation, and hematopoietic cell transplantation comorbidity index, conditioning intensity were well balanced between each group. The two-year OS of hCBT was equivalent to UR-BMT (hCBT: 53.6%, UR-BMT: 58.9%, p=0.56) (Figure 1). There was no significant difference in the two-year NRM between the two groups (hCBT: 24.9%, UR-BMT: 25.4%, p=0.87). The hCBT group had a lower neutrophil engraftment rate achieved at day 28 (hCBT: 83.0%, UR-BMT: 92.3%, p<0.001). The two-year relapse rate was similar between the hCBT and UR-BMT groups (hCBT: 26.1%, UR-BMT: 18.7%, p=0.34). The cumulative incidence of acute GVHD tended to be lower in the hCBT group (hCBT: 18.2%, UR-BMT: 33.6% at 100 days, p=0.067), and the same results were observed when analyzing grade 2-4 or grade 3-4 acute GVHD. The cumulative incidence of chronic GVHD was significantly lower in the hCBT group (hCBT: 4.7%, UR-BMT: 48.3%, p<0.001), and the cumulative incidence of severe chronic GVHD was also lower in hCBT group (hCBT: 0.0%, UR-BMT: 13.9%, p<0.001). To reduce the impact of patients' characteristics, propensity score matching was conducted. The covariates included prior allo-HCTs, disease risk index, and allo-HCT conducted before 2018 or not. After propensity score matching, a total of 116 patients were included in the analysis with 58 patients for each group. The two-year OS of hCBT was also equivalent to UR-BMT (hCBT: 61.8%, UR-BMT: 55.2%, p=0.43) (Figure 2). The two-year NRM was slightly lower in hCBT group, but it is not significant (hCBT: 19.1%, UR-BMT: 30.1%, p=0.21). The hCBT group had a late neutrophil engraftment (hCBT: 87.0%, UR-BMT: 89.1% at day 28, p<0.001). The two-year relapse rate was similar between the hCBT and UR-BMT groups (hCBT: 25.0%, UR-BMT: 20.0%, p=0.54). The cumulative incidence of acute GVHD was tended to be lower in hCBT group (hCBT: 19.8%, UR-BMT: 38.7% at 100 days, p=0.057) and the cumulative incidence of chronic GVHD was significantly lower in hCBT group (hCBT: 5.3%, UR-BMT: 56.3%, p<0.001). Discussion: This was the first study to validate that the outcomes of CBT with higher TNC were equivalent to UR-BMT. Although the hCBT group experienced later neutrophil engraftment, the rate of neutrophil engraftment achieved at day 28 was comparable to UR-BMT, and a lower incidence rate of chronic GVHD might be attributed to lower NRM. From these findings, hCBT may be a worth considering option for patients requiring allo-HCT.

The Relationship between Occurrence of aGVHD in Patients with Acute Myeloid Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation and Immune Cell Components in Graft

To explore the relationship between occurrence of acute graft-versus-host disease (aGVHD) and various immune cell composition in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 104 patients with AML undergoing allo-HSCT in our hospital were retrospectively analyzed, and the hematopoietic reconstitution and occurrence of GVHD were analyzed. Flow cytometry was used to detect the proportion of various types of immune cells in the grafts, the number of graft composition in patients with different degrees of aGVHD was calculated and compared, and to analyze the correlation between the severity of aGVHD in AML patients after allo-HSCT and the immune cell components in the graft. There was no significant difference in the time of hematopoietic reconstitution between the high number group of total number of nucleated cells (TNC) and the low number group, while the time of neutrophil and platelet reconstruction in the high number of CD34 group was significantly faster than that in the low number of CD34 group (P＜0.05), and the total hospital stay also tends to be shorten. Compared with patients in 0-Ι aGVHD group, both HLA-matched and HLA-haploidentical transplantation, the infusion amounts of CD3+ cells, CD3+CD4+ cells, CD3+CD8+ cells, NK cells and CD14+ monocytes were higher in patients of Ⅱ-Ⅳ aGVHD group， but the difference was not statistically significant (P>0.05); In addition, in patients with HLA-haploidentical transplantation, the number of CD4+CD25+ cells in Ⅱ-Ⅳ aGVHD group was significantly lower than that in 0-Ι aGVHD group (P<0.05), and the same trend was also observed in HLA-matched transplanted patients, but the difference was not significant (P=0.078). High number of CD34+ cells in the graft is beneficial to hematopoietic reconstitution in AML patients. To a certain degree, high number of CD3+ cells, CD3+CD4+ cells, CD3+CD8+ cells, NK cells and CD14+ cells tend to increase the occurrence of aGVHD, but high number of CD4+CD25+ regulatory T cells is beneficial to reduce the incidence of aGVHD in AML patients.

Validation of long-term handling and storage conditions for hematopoietic stem cell products for autologous transplants.

Hematopoietic stem cells (HPSCs) are multipotent stem cells that can differentiate into lymphoid and myeloid progenitors, giving rise to white blood cells (WBCs), red blood cells (RBCs), and platelets. HPSCs are a widely used treatment for many hematological non-malignant and malignant disorders. HPSCs can be used in the fresh or cryopreserved state for future use. Fresh HPSCs are typically stored at 2-6°C for up to 72 hours and are primarily used for allogeneic transplants or autologous transplants in myeloma and lymphoma patients. However, in some cases of autologous donations, HPSC transplantation is delayed more than three days after collection. In such situations, the cells are thawed after short-term preservation, resulting in a 35% cell viability loss. This study aimed to investigate the quality of HPSCs products after long-term storage exceeding 72 hours. The quality of HPSCs products was assessed by measuring viable CD34+ cell count, the total number of nucleated cells (TNC), and HPSCs recovery after different storage intervals of up to 120 hours in hypothermal storage. The mean total cell viability decreased by 2.18% within 72 hours and 7.4% within 120 hours, while mean CD34+ cell recovery was 92.61 % at 72 hours and 83.83 % at 120 hours in hypothermal storage. The mean TNC recovery was 89.93% at 72 hours and 76.18 % at 120 hours. All products were free from bacterial contamination for up to 120 hours under hypothermal storage conditions.

Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness.

Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38-Lin- cells (p &lt; 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p &lt; 0.01). CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.

EVALUATION OF PREDICTABILITY OF BONE MARROW CELL CELLULARITY FROM RELATED AND UNRELATED DONORS

Introduction: For successful hematopoietic stem cell transplantation, a minimum number of total nucleated cells should be obtained (TNC) during the bone marrow (BM) harvest procedure. Objectives: Evaluate the predictability of the BM TNC collected from an interim sample collected during the procedure and the factors related to high-cellular harvest. Methodology: This is a retrospective observational study including BM donors and recipient from 2017 to 2019. The final TNC was based on an interim quantification of TNC and was compared with the actual final TNC obtained. Results: 81 donors were included and interim TNC of 53 donors were available. Based on linear regression, a significant correlation was found between the volume of BM collected and the interim and final TNC (n=53; R2=0.83; P&lt;0.001). The relationship between donor and recipient weight significantly influenced the collection yield. There was also a positive correlation between the volume of BM collected and the interim and final count of TNC (n=53; r=0.88; p&lt;0.001). The difference in donor and recipient weight also had a positive correlation (p&lt;0.02). Conclusion: Our results showed that interim TNC quantification can help to achieve a better performance during the procedure, allowing real-time re-estimation of the volume needed to be harvested.

The 48th Annual Meeting of the European Society for Blood and Marrow Transplantation: Physicians - Poster Session (P001 - P578).

Background: Bone marrow donation is a procedure that takes place in a hospital operating room. In many hospitals, autologous red cell units are collected from donor before the harvest and reinfused immediately after the donation to reduce the risk of anemia. The blood donation stimulates hematopoiesis, so it's possible that autotransfusion has an impact on number of collected cells. The aim of this study is to assess whether blood donation before the harvest has an impact on the total number of nucleated cells in the product. Methods: Study design: Most of the donors undergo preharvest autologous blood collection. The procedure was not carried out if the weight difference is greater than 30kg in favor of the donor. Until March 2020, due to COVID-19, routine blood donations for autotransfusion were suspended. This situation gave an opportunity to assess, if the donation of blood has an impact on the efficiency of BM collection and hemoglobin level in donors' blood after harvest. Bone marrow was aspirated under general anesthesia, from both pelvic bones. According to the local protocol the volume of marrow and WBC was measured during donation;if the total number of nucleated cells was sufficient, the donation was finished. The collection was also completed, if the total volume of BM reached 15 ml per kg of donor's body weight. Laboratory analyses: The blood samples for morphology analyses were taken in the day of qualification, one day before and one day after harvest. The samples of BM were taken during donation to measure number of lymphocytes. Statistics: TheMann-Whitney U-test was used to determine the significance between the cohorts. Differences with p values <0.05 were considered as statistically significant. Results: Between March and October 2020, 15 BM donations from 15 healthy donors (10 men and 5 women, median age: 35 years, range 20-49 years) were performed;these donors were not referred for autotransfusion. The control group consisted of 34 donors, who underwent harvest between January 2019 and March 2020 (23 men and 11 women, median age 25 years, range 18-41 years). Total volume collected BM was comparable in both groups (median: 1182 ml in first vs 1277 ml in control group). The level of hemoglobin on the day of qualification was similar in both group: median 15.2 g/dL for donors who had blood donation;and 15.4 g/dL, for donors not referred to autotransfusion. One day before harvest, the first group had statistically lower level of hemoglobin (median 14.1 g/dL vs 14.8 g/dL, p < 0.001), however one day after BM collection both groups were comparable (median 11.1 g/dL vs 11.5 g/dL). Total number of nucleated cells in the product was comparable in both groups: median 204 × 108 in donors referred for autotransfusion vs 219 × 108 in control group. Conclusions: Autologous blood transfusion had no impact on Hb levels in blood after BM donation and had no impact on the efficiency of BM harvest. This does not support the routine use of autologous blood transfusion for unrelated BM donors.

A Systematic Review Comparing Bone Graft Harvest From the Iliac Crest, Tibia, and Calcaneus Donor Sites in Foot and Ankle Surgery

Category: Basic Sciences/Biologics; Other Introduction/Purpose: Bone autografts are frequently harvested for use in foot and ankle surgery. The gold standard harvest site is the iliac crest; however there is known morbidity with this harvest site. Alternative harvest sites include the tibia and calcaneus which are easily accessible for lower extremity surgery. Controversy exists regarding the osteogenic potential of autografts from each location. We performed a systematic review of the known data on cellular autografts from the iliac crest, tibia, and calcaneus focusing on the total cells harvested from each site as well as the presence of the critically important osteogenic osteoprogenitor cells. We also reviewed autograft volume, autograft consistency, surgical techniques, and the morbidities and complications associated with each bone autograft harvest location. Methods: In accordance with the PRISMA 2020 guidelines, a literature search of the PubMed, Cochrane Library, Web of Science, and Google databases from inception through January 2022 was performed. The search terms included: 'autograft,' 'bone graft,' 'foot and ankle surgery,' 'iliac crest,' 'tibia,' 'calcaneus,' reamer irrigator aspirator,' 'RIA,' 'mesenchymal cells,' 'osteoprogenitor cells,' 'quantity,' 'number,' 'amount,' 'yield,' 'concentration,' 'surface antigens,' 'surface markers,' 'CD markers.' Studies were included if autologous bone graft was harvested from the iliac crest, femur, tibia, or calcaneus and the cells in the harvest underwent histologic evaluation, quantification, or CD surface antigen identification. We included studies with bone graft as well as bone marrow aspirates used as samples. The nucleated cell and osteoprogenitor cell yield was recorded from each location; and MSC CD markers present at each harvest site were extracted from the eligible studies. Results: 13 studies met our inclusion criteria. Eight studies performed cell quantification from the harvest sites. Notably these studies only provide counting of total nucleated cells without specific identification of the osteoprogenitors. Colony-forming units were also reported and their findings are reviewed. Total cellular material was on average greatest in the iliac crest, with intermediate counts in the proximal tibia, and lowest in the calcaneus autograft samples. Five studies identified mesenchymal stromal cell (MSC) cluster of differentiation (CD) antigens at the harvest sites of interest. CD markers confirm osteoprogenitor cells are present in each sample location. Total number of osteoprogenitor cells remains unknown, however authors have attempted to extrapolate this number based on number of total nucleated cells and other data. Conclusion: Osteoprogenitor cells can be harvested from the iliac crest, tibia, and calcaneus. Greater total nucleated cell yields were identified in harvests from the iliac crest with descending amounts moving location distally in the limb. Nucleated cells were counted to provident estimates of osteoprogenitor cells yields in most studies, with flow cytometry confirmation that osteoprogenitors are present. Strict quantification of osteoprogenitor cells remains unknown. Multiple studies compared cellular material from bone marrow aspirates versus bone graft harvests themselves. Studies with modern techniques are needed to identify the true number of osteoprogenitors in each location.

The Effect of Malnutrition on T-cell Circadian Rhythms

Abstract In mammals, T-cell migration is under circadian control, likely to anticipate daily rhythms in infection risk. Glucocorticoids are a major controller of circadian processes and malnutrition is associated with increased glucocorticoid secretion. Previous studies suggest malnutrition may impart a “super-quiescent” phenotype to T-cells, enabling a greater number of naïve T-cells to survive short-term malnutrition albeit with diminished function. Thus, we hypothesize that malnourished T-cells may conserve energy by disengaging from rhythmic migration under circadian control and/or foregoing migration to reside in the bone marrow instead. To test this hypothesis, the total number of nucleated cells and naïve CD4+ and CD8+ T-cells in the blood, spleen, bone marrow, and brachial and mesenteric lymph nodes were enumerated by flow cytometry every four hours over the course of one day from control and malnourished mice. Additionally, expression levels of CD127 and CXCR4 in both T-cell populations and the concentration of glucocorticoids in the blood were assessed. A better understanding of how malnutrition affects the circadian rhythm of T-cell migration will not only help identify the mechanisms of how circadian rhythms work, but also how organisms’ circadian rhythms change in response to malnutrition. This knowledge of how malnutrition disrupts the circadian rhythm of T-cells may help improve vaccination strategies in malnourished children. Supported by NSF-MRI [DBI- 1920116] NSF -RUI [IOS-1951881]

Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco

IntroductionAn important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability.MethodsA total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital.ResultsSeven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 10⁶/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 10⁶/kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073).ConclusionLeukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

Hematopoietic Progenitor Cell Donation from Healthy Female Donors During Pregnancy: A Report of 10 Cases

The golden rule when collecting hematopoietic progenitors (HPs) from healthy volunteers is "donor safety." Pregnancy is an absolute contraindication for HP collection from unrelated donors; however, collection from a related pregnant donor is sometimes considered based on the urgency of the indication for transplantation and the available alternatives. Data on the safety and efficacy of this practice are limited. We conducted a retrospective chart review of an institution's transplantation database to characterize the safety and efficacy of HP donation from pregnant donors. Ten cases of HP donation from pregnant donors were identified, including 6 bone marrow grafts and 4 peripheral blood stem cell grafts. The median age of donors was 27.5 years. The median volume of the collected product was 521 mL (range, 128 to 1160 mL), the median number of total nucleated cells (TNCs) in the graft was 252×108 (range, 30.5 to 794×108), the median TNC concentration in the graft was 37×106 per mL (range, 4.7 to 214.6×106 per mL). The median number of CD34 cells in the graft was 142×106 (range, 6 to 763×106), and the median CD34 concentration in the graft was 20×104 per mL (range, 2 to 206×104 per mL). There were no safety issues or signals related to the procedure. HP collection from pregnant donors is relatively safe. This case series provides valuable information for practicing transplant physicians on how to counsel pregnant donors when this scenario is encountered in clinical practice.

Assessing the Immune Tumour Microenvironment (iTME) Using Multiplex Immunoflourescence Histochemistry (mIHC) Demonstrates Close Proximity of Cytotoxic T-Cells to Plasma Cells (PC) in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Aim: A dysfunctional iTME facilitates disease progression in MM. Studies have demonstrated the association between the spatial distribution of immune cells and progression of various cancers. Using mIHC we aim to describe quantitative and qualitative changes in CD3+CD8+ T-cells (T cytotoxic) in patients with MGUS, ND and relapsed/refractory MM (RRMM) and assess spatial proximity to PCs.Method: Formalin-fixed, paraffin-embedded trephine sections from pts with MGUS (n=32), NDMM (n=65) and RRMM (n=59) were sequentially stained for CD138, CD3, CD8 and checkpoint receptors (CPs) Tim3, Lag-3 and PD-1 (Figure 1). Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of T cytotoxic populations and analysis of proximity to PCs. Descriptive statistics and ordinary one-way ANOVA were applied as appropriate.Results: Patient demographics, disease characteristics, treatment (including prior therapies, where applicable), best response, duration of response, median progression free (PFS) and overall survival (OS) will be presented for all cohorts. There was no difference in BM cellularity or total number of nucleated cells assessed across the cohorts (p=0.16 and p=0.25). PC % was higher in the ND and RRMM compared to MUGS cohort (p<0.001). The average distance between T cytotoxic and PCs was similar between the cohorts (p=0.38), but a higher proportion of T cytotoxic were within 50μm of a PC in the ND cohort (p=0.0036, 90.8±15.8% (ND) vs. 77.6±19.5% (MGUS) and 80.1±25.9% (RR)). The % of unique PCs with a single T cytotoxic within 100μm is higher in patients with MGUS and RRMM than NDMM (p=0.0007). There was no difference in the %CD3+, %CD3+CD8+ or %CD3+ cells expressing CD8 (p=0.22, p=0.62, p=0.48). CP expression on T cytotoxic was similar (Tim3 p=0.46, Lag-3 p=0.35; PD-1 p=0.54) with no difference in dual or triple CP expression. Sub-analyses assessing CP expression patterns and T cytotoxic/PC proximity within individual cohorts based on response to treatment/disease progression are to follow.Conclusion: The infiltration of cytotoxic T cells into tumours is a critical factor in immunotherapy efficacy. Here we clearly demonstrate the feasibility of mIHC to describe the spatial context of the iTME and we plan to implement it for predictive value in future studies of immunotherapies in patients with MM.Figure 1: Bone marrow FFPE trephine section stained with mIHC using the Opal TM workflow demonstrating plasma cells (CD138, green), T cells (CD3, yellow; CD8, pink) and checkpoint receptors (PD-1, orange; Lag-3, magenta; Tim3, light blue) with colocalisation of some signals. DisclosuresHarrison: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/ Juno/ BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eusa: Consultancy, Honoraria, Speakers Bureau; Terumo BCT: Consultancy, Honoraria. Quach: Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Tumor Infiltrating Lymphocytes (TILs) and T Cell Fraction in Human Papillomavirus (HPV) Associated Oropharyngeal Squamous Cell Carcinoma

Purpose/Objective(s)The percentage of tumor infiltrating lymphocytes (TILs) is indicative of immune response and prognostic in many malignancies, although TILs data within HPV associated oropharyngeal cancer are limited. Our prior analyses of HPV associated malignancy revealed that both a higher fraction of T cells (TCF) to total cells present in the primary tumor specimen and a higher percentage of TILs were associated with a decreased chance of cancer recurrence. Herein, we investigated the relationship between these two immune related prognostic factors, TILs and TCF.Materials/MethodsPatients had HPV(+) oropharyngeal cancer and underwent primary surgery between 2007 and 2016. One representative H&E slide of the primary tumor was independently reviewed by two pathologists blinded to outcome for TILs count and presence/absence of desmoplastic stroma. The Brandwein-Gensler pattern of invasion (POI) score was used to grade the primary tumor. For TCF, immunosequencing of the CDR3 regions of human TCRβ chains was performed using an immunosequencing assay. TCF was determined as the proportion of T cells compared to the total number of nucleated cells in a sample. The TCF for patients by primary tumor TILs density, desmoplastic stroma, and POI were evaluated using Wilcoxon rank sum tests.ResultsTwenty-four patients were identified who had both TCF and TILs count available for comparison. Median age was 55 years (IQR 47-61), 96% were male, 42% had a > 10 pack year history of smoking, and 83% underwent adjuvant therapy. For patients with < 10% TILS, median TCF was 0.29 (IQR 0.17-0.36) as compared to TCF of 0.40 (IQR 0.34-0.57) for patients with ≥10% TILs (P = 0.09). There was no statistically significant difference (P = 0.5) in TCF for patients with desmoplastic stroma absent (median TCF 0.41; IQR 0.36-0.48) versus desmoplastic stroma present (median TCF 0.34; IQR 0.23-0.50). Patients with POI I or II had similar TCF to those with type III or IV, median TCF 0.37 (IQR 0.30-0.48) versus 0.35 (IQR 0.23-0.50), respectively (P = 0.8).ConclusionIn this series of HPV+ oropharyngeal cancer patients, TILs ≥10% was associated with a higher fraction of T cells to total cells (TCF) (n = 24, P = 0.09). Additional investigation is warranted including the relationship of TCF as characterized by sequencing and pathologist quantification of TILs, and whether the combination of these factors improves prognostication over each alone. The percentage of tumor infiltrating lymphocytes (TILs) is indicative of immune response and prognostic in many malignancies, although TILs data within HPV associated oropharyngeal cancer are limited. Our prior analyses of HPV associated malignancy revealed that both a higher fraction of T cells (TCF) to total cells present in the primary tumor specimen and a higher percentage of TILs were associated with a decreased chance of cancer recurrence. Herein, we investigated the relationship between these two immune related prognostic factors, TILs and TCF. Patients had HPV(+) oropharyngeal cancer and underwent primary surgery between 2007 and 2016. One representative H&E slide of the primary tumor was independently reviewed by two pathologists blinded to outcome for TILs count and presence/absence of desmoplastic stroma. The Brandwein-Gensler pattern of invasion (POI) score was used to grade the primary tumor. For TCF, immunosequencing of the CDR3 regions of human TCRβ chains was performed using an immunosequencing assay. TCF was determined as the proportion of T cells compared to the total number of nucleated cells in a sample. The TCF for patients by primary tumor TILs density, desmoplastic stroma, and POI were evaluated using Wilcoxon rank sum tests. Twenty-four patients were identified who had both TCF and TILs count available for comparison. Median age was 55 years (IQR 47-61), 96% were male, 42% had a > 10 pack year history of smoking, and 83% underwent adjuvant therapy. For patients with < 10% TILS, median TCF was 0.29 (IQR 0.17-0.36) as compared to TCF of 0.40 (IQR 0.34-0.57) for patients with ≥10% TILs (P = 0.09). There was no statistically significant difference (P = 0.5) in TCF for patients with desmoplastic stroma absent (median TCF 0.41; IQR 0.36-0.48) versus desmoplastic stroma present (median TCF 0.34; IQR 0.23-0.50). Patients with POI I or II had similar TCF to those with type III or IV, median TCF 0.37 (IQR 0.30-0.48) versus 0.35 (IQR 0.23-0.50), respectively (P = 0.8). In this series of HPV+ oropharyngeal cancer patients, TILs ≥10% was associated with a higher fraction of T cells to total cells (TCF) (n = 24, P = 0.09). Additional investigation is warranted including the relationship of TCF as characterized by sequencing and pathologist quantification of TILs, and whether the combination of these factors improves prognostication over each alone.

P-074: Assessing the immune microenvironment with multiplex immunofluorescence histochemistry demonstrates proximity of cytotoxic T-cells to plasma cells in patients with newly diagnosed multiple myeloma

<h3>Background</h3> A dysfunctional immune tumour microenvironment (iTME) facilitates disease progression in MM. Using multiplex immunohistochemistry (mIHC) we aim to describe quantitative and qualitative changes in CD3+CD8+ T-cells (Tcytotoxic) in patients with MGUS, ND and relapsed/refractory MM (RRMM) and assess spatial proximity to PCs. <h3>Method</h3> Formalin-fixed, paraffin-embedded trephine sections from pts with MGUS (n=32), NDMM (n=65) and RRMM (n=59) were sequentially stained for CD138, CD3, CD8 and checkpoint receptors (CPs) Tim3, Lag-3 and PD-1 (Figure 1). Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of Tcytotoxic populations and analysis of proximity to PCs. Descriptive statistics and ordinary one-way ANOVA were applied as appropriate. <h3>Results</h3> Patient demographics, disease characteristics, treatment (including prior therapies, where applicable), best response, duration of response, median progression free (PFS) and overall survival (OS) will be presented for all cohorts. There was no difference in BM cellularity or total number of nucleated cells assessed across the cohorts (p=0.16 and p=0.25). PC % was higher in the ND and RRMM compared to MUGS cohort (p<0.001). The average distance between Tcytotoxic and PCs was similar between the cohorts (p=0.38), but a higher proportion of Tcytotoxic were within 50μm of a PC in the ND cohort (p=0.0036, 90.8±15.8% (ND) vs. 77.6±19.5% (MGUS) and 80.1±25.9% (RR)). The % of unique PCs with a single Tcytotoxic within 100µm is higher in patients with MGUS and RRMM than NDMM (p=0.0007). There was no difference in the %CD3+, %CD3+CD8+ or %CD3+ cells expressing CD8 (p=0.22, p=0.62, p=0.48). CP expression on Tcytotoxic was similar (Tim3 p=0.46, Lag-3 p=0.35; PD-1 p=0.54) with no difference in dual or triple CP expression. Sub-analyses assessing CP expression patterns and Tcytotoxic/PC proximity within individual cohorts based on response to treatment/disease progression are to follow. <h3>Conclusion</h3> Multiplex IHC is a novel technique to assess the iTME. While there is no discernible quantitative difference in cytotoxic T-cells or checkpoint receptor expression, we demonstrate increased proximity of cytotoxic T-cells to plasma cells in newly diagnosed patients suggestive of a more robust immune system compared to multiply treated patients.

Bone Marrow Transplant Product Quality Has Decreased Over Time— a Retrospective Examination of NMDP Collected Bone Marrow Products from 1994-2016

▪BackgroundBone marrow (BM) is an essential hematopoietic stem cell (HSC) source for many allogeneic transplants (HCT) and is the preferred cell source for specific disease indications in adults (e.g., Aplastic Anemia), the majority of pediatric transplants, and when the benefits of a decreased risk of chronic graft versus host disease (GVHD) outweigh other considerations. The advent of G-CSF mobilized peripheral blood stem cells (PBSC), with logistic benefits over the collection of BM, has resulted in a significant decrease in the utilization of BM. Since the advent of PBSC, BM has declined from 100% in the early 1990s to 15% in 2015, of unrelated donor products. This decreased utilization of BM has a number of potential consequences from a quality perspective. Many centers perform a limited number of BM collections per year and thus, the ability to ensure ongoing competency of staff in this technical procedure can be challenging. Proper assessment of BM harvest metrics is difficult in an individual collection facility when limited procedures are performed. Previous data from a single center noted that the total number of nucleated cells (TNC) received in BM products for individual patients decreased over the years. The aim of this study was to examine the trends in BM harvest quality in a large cohort of NMDP donors to see if this observation can be generalized.MethodsThe primary outcome of this study was examination of the total nucleated cells collected per milliliter (TNC/mL) of BM as an estimate of product quality. The population studied was BM donations from domestic unrelated donors reported to the NMDP/CIBMTR from 1994-2016 (analyzed over 5 time periods). The TNC/mL for each harvest was calculated based on the total number of nucleated cells (TNC) collected in harvest and the volume (mL) of final product including additives. Additional covariates, including time period of harvest, the number of harvests performed per year at the center, donor age, donor body mass index (BMI), donor weight, donor race/ethnicity, and donor gender were examined.ResultsExamination of >15,000 BM donations revealed a significant decline in the concentration of total nucleated cells from 21.8 TNC x 106/mL in the earliest era to 18.7 TNC x106/mL in the 2012-2016 era (p<0.001). (Table 1). The number of overall collections performed per center in the 2012-2016 era was similar to other eras (Table 2). The number of collections performed by a collection center did not appear to explain the decrease in BM quality as median TNC/mL decreased over time intervals regardless of collection centre size (Figure 1). Interestingly, the total product volume harvest increased over the eras (p<0.001), while the duration of the harvest decreased (p<0.001). Donor characteristics were examined across eras. In recent years, donors are significantly more likely to be male (p<0.001) and young (p<0.001). For example, in the 2012-2016 era, 54% of the donors were under the age of 30 as compared to the 1994-1996 and 1997-2001 eras where only 25% of the donors were under the age of 30. Similarly, 65% of the donors were male in the 2012-2016 era, compared to 53% in the 1994-1996 era.ConclusionIn conclusion, we showed a significant decrease in the TNC/mL in BM products over time. This did not appear to be related to the number of collections per center, suggesting that cell number collected is not directly related to the number of procedures performed but rather other factors in the collection process. Interestingly, the significant decrease in TNC concentration in BM observed in this study persisted despite the increased use of optimal donors. Further analysis will be required to elucidate the exact cause of decreased concentration of TNC collected in products. Factors such as technique, rate of collection, materials /equipment used, training, and collector experience may have profound influences on the quality of BM collected. For example, the number of personnel performing harvests, number of harvests performed per collector, or turn-over of personnel at each collection center maybe contributing factors. These further studies will help to better understand the entire process and help improve the quality of the products, an issue crucial to ensuring bone marrow remains a quality cell source for transplant. [Display omitted] DisclosuresPulsipher:Jazz Pharmaceuticals: Other: advisory board, education; CSL Behring: Other: advisory board Feb 2017; Adaptive: Other: Advisory board 6/17; Chimerix: Other: Advisory board Dec 2015; Novartis Pharmaceuticals Corporation: Consultancy, Other: Phase II steering committee.

Outcomes of Unrelated Cord Blood Transplantation for Familial Hemophagocytic Lymphohistiocytosis: Risk Factors and Long Term Follow-up. an Analysis on Behalf of Eurocord, Cord Blood Committee of Cellular Therapy and Immunobiology Working Party of the EBMT and Inborn Errors Working Party of the EBMT

Familial Hemophagocytic Lymphohistiocytosis (FHLH) is a systemic hyperinflammatory syndrome caused by excessive activation of lymphocytes and macrophages that produce high levels of cytokines as a result of genetic defects affecting the granule-mediated cytotoxicity pathway. Allogeneic hematopoietic stem cell transplantation (HSCT) is so far the only available curative option for the disease and the majority of studies have been conducted in patients given either bone marrow or peripheral blood transplantation. Previous case series of unrelated umbilical cord blood transplants (UCBT) reported survival rates ranging from 25%-85%. We analyzed the outcomes of 118 children from 43 EBMT centers with HLH undergoing single UCBT and reported to Eurocord. The median year of UCBT was 2010 (range 1996-2014). The median follow-up was 75 months (range 3.3-211). The majority of patients had genetic diagnosis of familial HLH (64%) of which 37.4% were due to perforin defect, 30.6% due to Munc 13-4 defect, 25.3% due to Munc 18-2, 5.4% due to syntaxin 11 and 1.3% due to perforin and syntaxin 11 defects. The median age at diagnosis and at UCBT were 0.3 years (range 0-14) and 1 year (range 0.1-14), respectively. The median time from diagnosis to UCBT was 5.3 months (range 0-57). Amongst 113 patients with known disease status, most patients underwent UCBT in complete remission 68/113 (60%) with 25/113 (22%) in partial remission and 20/113 (18%) having active disease at the time of transplant. Conditioning regimen was Busulfan or Treosulfan-based myeloablative in 78% of the patients while 22% received a reduced intensity conditioning. Seventeen (14%) children did not receive serotherapy as part of conditioning. Thirty-five patients (30%) received a 6/6 HLA matched cord blood unit, 56 (48%) received a 5/6 HLA mismatched cord blood unit, 24 (21%) received a 4/6 (21%) and 1 patient received 3/6 (1%) HLA mismatched graft. The median number of total nucleated cells (TNC) infused was 9.9 x107/kg (range 1.26 - 42.5 x107/kg).The cumulative incidence (CI) of neutrophil engraftment was 85% (78%-92%) at 60 days with 79% of engrafted patients exhibiting full donor chimerism at day 100.The CI of grade II to IV acute graft-versus-host disease (aGVHD) was 33% (26%-43%) at day 100, while the 6-year CI of chronic GVHD was 17% (11%-26%). The CI of relapse and non-relapse mortality (NRM) at 6 years was 14% (9%-22%) and 36% (28%-46%), respectively.The estimated 6-year overall survival (OS) and disease-free survival (DFS) was 55% (45%-64%) and 50% (40%-60%), respectively. Fifty-two patients died: 10 of relapse and 37 of transplant related causes (the most frequent causes being: infections, n=8; GVHD n=8 and ARDS, n=4).In univariate analysis, children receiving more than 9.9x107/Kg infused TNC had higher rates of engraftment (90% versus 79%, p=0.02) and better OS (65% versus 42%, p=0.04) compared to those patients receiving less cells. Interestingly, a number of TNC more than the median (TNC>9.9x107/Kg) was also associated with decreased incidence of grade II-IV acute (25% versus 43%, p=0.05) and chronic GVHD (3% versus 32%, p<0.01). In multivariate analysis, a number of TNC >9.9x107/Kg was associated with improved OS (HR 0.48, 95%CI 0.27-0.88, p=0.02), greater engraftment rate (HR 1.80, 95%CI 1.16-2.80, p=0.009) and decreased incidence of chronic GVHD (HR 0.10, 95%CI 0.02-0.45, p=0.003).DFS at 6 years was 69% for a HLA matched (6/6), 49% for 5/6 and 34% for 2 or 3/6 and 6 years NRM was 23%, 39% and 45%, respectively. In multivariate analyses, a graft with more than 2 HLA mismatches was associated with worse DFS and higher NRM (HR 2.81, 95%CI 1.25-6.35, p=0.01 and HR 2.72, 95%CI 1.02-7.23, p=0.04, respectively). We did not observe an impact of disease status at the time of transplant on OS, DFS, relapse incidence and NRM. There was no difference between the use of reduced intensity versus myeloablative conditioning regimen or using serotherapy on the study outcomes. No risk factors were found to be associated with relapse incidence. In conclusion, UCBT for FHLH with a higher cell dose and a well HLA matched cord blood unit (6/6 or 5/6) is a suitable alternative stem cell source for children who lack an HLA- matched BM or PBSC donor. Further studies are warranted to establish the best conditioning regimens to improve outcomes with reduced toxicity and to compare outcomes after UCBT with other alternative approaches such as unrelated donors and haploidentical HSCT. DisclosuresNo relevant conflicts of interest to declare.

The Influence of Low- and High-Negative-Pressure Liposuction and Different Harvesting Sites on the Viability and Yield of Adipocytes and Other Nucleated Cells

The volume effect of fat grafting is highly dependent on the presence of viable adipocytes and other nucleated cells within the lipoaspirate. We suspected that one of the crucial factors influencing cell viability is the negative pressure applied during the fat graft harvesting and the suitability of various harvest sites when compared to others. Despite much discussion, there is no consensus on the optimal negative pressure or the best site for harvesting so we designed an experiment to test this. Fat graft taken under low negative pressure (- 200 mmHg) or high negative pressure (- 700 mmHg) from the thigh or abdominal regions from 21 healthy human donors was evaluated. The principal variables studied were: a) total number and viability of nucleated cells, b) liposuction duration and c) blood admixture. Other variables studied were body mass index, the impact of age and enzymatic digestion. The absolute number and viability of nucleated cells and the blood admixture did not differ significantly between lipoaspirates obtained under different vacuum conditions or from different regions. The time taken to acquire the same volume of lipoaspirate was significantly increased using low negative pressure. The time taken to collect cells in the thigh region significantly increased with increasing BMI but this correlation was not found when harvesting in the abdominal region. The BMI and age did not impact the results in any of the measured variables. The enzymatic digestion rate was independent of the negative pressure used to harvest. Our results indicate that neither the negative pressure used nor the area chosen has any significant influence on the viability and yield of harvested cells. The time taken to obtain lipoaspirate using low pressure is significantly longer than when using high pressure. No significant difference was found in the value of blood admixture using different vacuum pressures, and no correlation exists between the body mass index and the cell viability or age of the patients and the time of liposuction. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266 .

In vitro expansion of fetal liver hematopoietic stem cells

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Verbascoside alleviates renal fibrosis in unilateral ureteral obstruction rats by inhibiting macrophage infiltration.

Objective(s): To explore the effect of verbascoside on renal fibrosis in unilateral ureteral obstruction (UUO) rats.Materials and Methods:Twenty Sprague-Dawley rats were randomly distributed into sham-operated, UUO, and UUO+Verbascoside groups. After two weeks of rat model construction, urine and blood samples were collected for biochemical analysis while kidney tissues were harvested for hematoxylin and eosin (H&E), Masson’s Trichrome, and immunohistochemistry staining. Pearson coefficient was used to analyze the correlation between the two proteins. Results:Verbascoside improved UUO-induced renal dysfunction as detected by decreased serum creatinine, urea nitrogen, and urinary protein excretion rate. In UUO rats, H&E staining result revealed increased total nucleated cell number, and Masson’s Trichrome staining results showed tubular interstitial fibrosis with the deposition of collagen fibrils. Besides, expressions of fibrosis-related proteins including collagen type I (COL-I), α-smooth muscle actin (a-SMA), and tissue inhibitor of metalloproteinase 2 (TIMP2) expressed higher in the UUO group. Moreover, macrophage infiltration-related factors such as CD68+, F4/80+ cells, and suppressor of cytokine signaling-3 (SOCS3) were significantly higher in the UUO group than in sham-operated rats. However, after administration with verbascoside, the accumulation of collagen fibrils and total nucleated cell numbers were mitigated. Likewise, macrophage infiltration was extenuated and fibrosis-related proteins were down-regulated in the UUO+Verbascoside rats. Correlation analysis indicated that macrophage infiltration-related markers were related to fibrosis-related factors.Conclusion:Verbascoside could alleviate renal fibrosis in UUO rats probably through ameliorating macrophage infiltration.