Total Nucleated Cell Concentration Research Articles

Factors Associated with Increased Risk of Contamination in Bone Marrow Transplants.

Hematopoietic cell transplantation (HCT) remains the definitive therapeutic modality for numerous malignant and non-malignant hematologic disorders. Conventional bone marrow remains a viable donor source for HCT. However, microbial contamination of bone marrow harvests may present a risk to immunocompromised recipients. This analysis sought to identify clinical factors associated with bone marrow contamination. We analyzed a single institution experience comprising 667 unique bone marrow harvests collected between 1999 and 2021. We trended the yearly microbial contamination rate over this time span. Harvest type (autologous vs allogenic), donor age, donor sex, physician experience, total nucleated cell (TNC) count, volume collected, and TNC concentration were included in a univariate (UV) and multivariate (MV) logistic model to assess which factors were associated with contamination. Males comprised 55.8% of the donor population and the median age of the cohort was 35 [IQR: 27-45]. There were 19 autologous, 151 related allogenic, and 497 unrelated allogenic transplants in this cohort. 87 of the 667 (13.0%) harvests were contaminated and essentially all contaminants were common skin flora. The yearly contamination rates displayed substantial variability, ranging from 0% to 42.9%, with no discernible trend. Harvest type did not exhibit a significant association with contamination risk. However, male donor sex was found to be significantly associated with a higher contamination rate (18%) compared to female sex (6.8%, p < 0.001). In both UV and MV logistic models, male sex emerged as the sole factor linked to contamination risk (OR: 2.90, 95% CI: 1.65 - 5.35). This analysis represents the largest single center investigation of bone marrow harvest microbial contamination rates. Notably, it is the first to establish an association between contamination and male donor sex. We propose that this association may be attributed to differences in skin flora composition or innate immune function between sexes, general hygiene practices or possibly the result of the frequent clipping of body hair in males prior to the harvest procedure. Further research is warranted to explore the underlying mechanisms and clinical implications of this novel finding.

Quantification of the cellular content of platelet-rich plasma harvested after injection of filgrastim versus pegfilgrastim biosimilars: a prospective, single-center, crossover study

IntroductionHematopoietic stem cells (HSCs) appear in peripheral blood (PB) following mobilization due to external events or pharmaceutical agents like filgrastim and pegfilgrastim. Concentration of these into platelet-rich plasma (PRP) can be used in orthopedics. ObjectivesThis prospective, single-center, controlled laboratory study aimed to compare safety profiles of filgrastim and pegfilgrastim administration, quantifying the cellular content of PRP derived from PB post-administration. We hypothesized comparable rates and severity of adverse events (AEs) among participants, along with similar cellular content in the PRP products. MethodsTen healthy male participants underwent a crossover design with 2 interventions separated by a washout period. PB was collected before and after each intervention, producing PRP for cellular comparison in vitro. AEs were monitored for severity and frequency throughout study. ResultsSignificant AEs for filgrastim included fatigue (36%), myalgia (36%), and back pain (9.1%), compared to pegfilgrastim, which included myalgia (31%), fatigue (23%), and back pain (23%). There were no significant differences between AE occurrences (P = .49) nor relationship to study drug (P > .99). Regarding total nucleated cell count (TNC), statistical significance was found in TNC count (P = .010) and TNC concentration (P = .004). All PRP products expressed high levels (>88%) of hematopoietic progenitor cell/HSC-specific CD45+, CD34+, CD45dim+, and hematopoietic progenitor cell/HSC+ markers. ConclusionsThis study demonstrated that filgrastim and pegfilgrastim administration appear safe. Pegfilgrastim is equivalent to filgrastim for the mobilization of white blood cells and cells expressing hematopoietic cell-surface markers into the PB and consequently, into the derived PRP product. Clinical relevanceThis study presents pegfilgrastim as a single-injection alternative to filgrastim for the mobilization of HSCs into the PB and subsequent PB-derived PRP, which is more convenient and less invasive for patients.

Failure and out of Specification Manufacturing of Autologous CAR-T Cells Could be Associated with a High Concentration of Total Nucleated Cells, CD3 + Cells and Neutrophils in the Apheresis Product

Manufacturing capacities of commercial autologous chimeric antigen receptor (CAR)-T cells have been significantly increased during the last years, allowing treatment of a larger patient cohort. However, this was associated with the emergence of several failures and out of specifications (OOS), resulting in treatment delay. The aim of our study was to identify early indicators related to apheresis products that can predict CAR-T cells manufacturing failure or OOS. To this end, we analyzed manipulation processes of cellular starting materials (conservation, freezing, cryopreservation) and quality controls (QC) including count and/or concentration of total nucleated cells (TNC), CD3 +, CD4 +, CD8 + cells, platelets, monocytes and neutrophils. We analyzed 802 apheresis products that were collected for the manufacturing of axicabtagene ciloleucel (axi-cel, n=303), tisagenlecleucel (tisa-cel, n=403), brexucatagene autoleucel (brexu-cel, n=44) and idecabtagene vicleucel (ide-cel, n=52). This cohort was classified into 3 groups: failure (CAR-T cells not produced), OOS (CAR-T cells produced but non-compliant) and compliant. We identified 21 failures and 24 OOS, resulting in either no treatment or a significant delay when the OOS were injected (57.6±17.5d versus 45.9±16.3d in compliant group, p=0.0405). Among each CAR-T cells product, 13 (4.3%) axi-cel, 30 (7.4%) tisa-cel, 1 (2.3%) brexu-cel and 1 (1.9%) ide-cel were concerned. The main causes of failures were low viability (33.3%), non-compliant dose (28.6%), microbiological contamination (23.8%) and formulation concerns (14.3%). In OOS, low viability (41.7%), low dose (29.2%), microbiological contamination (4.2%), formulation (12.5%) and safety concerns (8.3%; i.e high vector copy number) were identified. 17 products (13 failures and 4 OOS) were successfully remanufactured without an increased delay comparing to compliant products (47.5±15.9 and 47.3±15.1 respectively versus 45.9±16.3 d). To identify risk factors of manufacturing failure or OOS due to non-compliant viability and/or dose, we analyzed processing and QC of collected cellular starting materials. OOS were associated with a high concentration of TNC (146.7±94.3 vs 61.6±36.1 x10 6/mL, p=0.009) and CD3 + cells (88.6±76.5 vs 26.0±21.9 x10 6/mL, p=0.0112) in cell collections, while failures were observed in apheresis with high neutrophils concentration (14.3±15.03 x10 3/µL vs 6.0±7.6 x10 3/µL, p=0.0195). Interestingly, neutrophils were significantly decreased in apheresis collected for CAR-T cells remanufacturing which succeed (p=0.0156). This preliminary study showed that high TNC, CD3 + cells and neutrophils concentration in the apheresis product might be predictive factors of failures and/or OOS of commercial autologous CAR-T cells manufacturing.

What is your diagnosis? Peritoneal fluid from a dog.

What is your diagnosis? Peritoneal fluid from a dog.

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.

Cerebrospinal fluid analysis lacks diagnostic specificity in dogs with vestibular disease.

Although, vestibular syndrome is a common neurological presentation, little is known about the diagnostic value of cerebrospinal fluid (CSF) analysis in vestibular syndrome in dogs. Medical records were retrospectively reviewed, and dogs with vestibular disease that had undergone magnetic resonance imaging of the head, CSF analysis and were diagnosed with central or peripheral vestibular syndrome were included. Disorders affecting the central vestibular system included meningoencephalitis of unknown origin (MUO), brain neoplasia, ischaemic infarct, intracranial empyema or metronidazole toxicity. Disorders affecting the peripheral vestibular system included idiopathic vestibular disease, otitis media/interna or neoplasia affecting the inner ear structures. Total nucleated cell concentration (TNCC), total protein concentration (TP) and cytologic assessment were recorded. A total of 102 dogs met the inclusion criteria. The sensitivity and specificity of increased CSF TNCC to differentiate central from peripheral vestibular syndrome was 49% and 90%, while the sensitivity and specificity of increased TP was 58% and 39%, respectively. The TNCC and TP in dogs with MUO were significantly higher than in dogs with idiopathic vestibular disease (p=0.000 and p=0.004). MUO was associated with lymphocytic pleocytosis, while idiopathic vestibular disease and ischaemic infarct were associated with the presence of activated macrophages or normal cytology (p=0.000). Although consistent CSF abnormalities were observed in dogs with MUO, CSF analysis did not allow reliable differentiation between central and peripheral vestibular syndrome. CSF analysis is not reliable as the sole diagnostic technique in dogs with vestibular disease.

Shorter Interdonation Interval Contributes to Lower Cell Counts in Subsequent Stem Cell Donations

Approximately 7% of unrelated hematopoietic stem cell donors are asked to donate stem cells a subsequent time to the same or a different recipient. Recent studies have shown that donation-related symptoms for second donations are similar to those for the first donation. Little is known about differences in stem cell mobilization and yields for subsequent peripheral blood stem cell (PBSC) and bone marrow (BM) collections. We hypothesized that CD34+ cell yields and total nucleated cell (TNC) concentrations for subsequent PBSC or BM donations are lower than those at the first donation. We also evaluated the factors influencing stem cell yields in healthy unrelated second-time donors. Data were gathered from the Center for International Blood and Marrow Transplant Research database on 513 PBSC and 43 BM donors who donated a second time between 2006 and 2017 through the National Marrow Donor Program. Among the second-time PBSC donors, we found significantly lower preapheresis peripheral blood CD34+ cell counts (68.6×106/L versus 73.9×106/L; P=.03), and collection yields (556×106 versus 608×106; P=.02) at the second donation compared to the first. This decrease at the subsequent donation was associated with a shorter interdonation interval, lower body mass index (BMI), and a lower total G-CSF dose. In most instances, suboptimal mobilizers at their first donation donated suboptimal numbers of stem cells at their subsequent donations. Among repeat BM donors, the TNC concentration was lower at the second donation. The small size of this group precluded additional analysis. Overall, when considering repeat donations, increasing the interdonation intervals and evaluating for BMI changes should be considered to optimize stem cell yields. Some of these parameters may be improved by increasing G-CSF dose in PBSC donors within permissible limits.

Cellular and Clinical Analyses of Autologous Bone Marrow Aspirate Injectate for Knee Osteoarthritis: A Pilot Study.

Knee osteoarthritis (OA) is characterized by pain and functional deficits. Common conservative strategies include medications, physical therapy, and intra-articular injections. Recently, treatment using autologous cell injections has increased. To characterize the cellular content of bone marrow aspirate (BMA) and to evaluate the effect of intra-articular autologous BMA injections in patients with mild knee OA. Prospective pilot observational study. Academic institution. Eleven patients with unilateral or bilateral mild knee OA (15 knees) were included in the cellular analysis. Ten patients (13 knees) were included in the overall (cellular and clinical) analysis. BMA was aspirated from patients' iliac crests and then injected intra-articularly under fluoroscopic and/or ultrasound guidance. BMA samples were analyzed using flow cytometry, colony forming unit (CFU) assays, and enzyme-linked immunosorbent assays. Questionnaires assessing pain and function were administered preinjection and at 1, 3, 6, and 12 months postinjection. Side effects and satisfaction were assessed. Total nucleated cell (TNC) concentration, mesenchymal stem cell (MSC) concentration, CFU count, and interleukin-1 receptor antagonist (IL-1Ra) concentration. BMA sample analyses revealed wide ranges in TNC concentration (173300-4 491 050 cells/mL), MSC concentration (0-500 cells/mL), CFUs (0-19), and IL-1Ra concentration (2806-29 394 pg/mL). Improvements in Knee Injury and Osteoarthritis Outcomes Score for Joint Replacement were observed throughout the 12-month follow-up period (F[4,12] = 12.29, P < .001). Additionally, current, usual, best, and worst numerical rating scale pain scores significantly decreased over time (P < .001). Patient satisfaction was high (range: 8.1 ± 2.1-8.8 ± 1.9), and side effects were uncommon. The cellular content of BMA samples varied widely between patients and was lower than the anticipated yield reported by the device's manufacturer. However, intra-articular BMA injections for knee OA in a small pilot cohort appeared to be safe with potential therapeutic value. Larger, prospective, double-blinded studies are warranted.

A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies

Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow–derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

Factors associated with blood cord unit bankability: an analysis of a 15-year-long case series.

Blood banking is a long and complex process requiring an accurate screening of potential donors and high-quality control systems. Previous studies in literature investigated factors potentially determining a higher cell levels with the aim of optimizing donors' selection and improving banking process. This study aims to identify factors associated with the concentration of stem cells in umbilical cord blood, so increasing the probability of bankability, focusing on the possible implications in terms of obstetric and resources management. This is a retrospective study conducted in the Obstetric Units of two Italian Hospitals in Montebelluna and Castelfranco Veneto. Study has been conducted on cord blood units banked between 1999 and 2015. Data on medical histories and clinical characteristics of mother and baby have been retrieved via a retrospective examination of medical records. A total of 869 cord blood units were studied. At multivariable analysis, in agreement with literature, birthweight and placental weight have been found to be associated with higher concentration of total nucleated cells. As additional factor, amount of fluid infused was associated with cord blood units' count. This study is the first one to clearly identify the role of fluid infusion on cord blood units' counts in addition to placental weight and delivery. Some non-modifiable features can help in predicting bankability from pre-natal aspects to factors more related with obstetric management is suggested.

Evaluation of concurrent analysis of cerebrospinal fluid samples collected from the cerebellomedullary cistern and lumbar subarachnoid space for the diagnosis of steroid-responsive meningitis arteritis in dogs.

To evaluate whether concurrent analysis of CSF samples from 2 collection sites (cerebellomedullary cistern [CMC] and lumbar subarachnoid space [LSS]) versus only 1 site could improve the diagnostic sensitivity of CSF analysis for dogs with suspected steroid-responsive meningitis arteritis (SRMA). 111 client-owned dogs with SRMA diagnosed at 3 veterinary referral hospitals between 2011 and 2017. Only dogs with CSF collected from both sites (CMC and LSS) and with no previous history of corticosteroid administration were included. Medical record data and logistic regression were used to identify factors associated with having a total nucleated cell concentration (TNCC) within the reference interval in a CSF sample from 1 collection site. The TNCC was within the reference interval (TNCC < 5 cells/μL) in the CSF sample from 1 collection site for 8 of 111 (7%) dogs and was only slightly high (TNCC, 5 to 9 cells/μL) in the sample from 1 or both sites for 10 (11%) other dogs. For each of these 18 dogs, results for samples from 1 site were consistent with SRMA. The proportion of CSF samples that had a TNCC within the reference interval was comparable between sites. As age increased, so did the risk of having an unremarkable TNCC in the CSF sample from 1 site, albeit only slightly (OR, 1.08; 95% confidence interval, 1.01 to 1.16). CSF samples from both the CMC and LSS should be analyzed when evaluating dogs with suspected SRMA to improve the chance of detecting a high TNCC.

Donor-matched comparison of chondrogenic progenitors resident in human infrapatellar fat pad, synovium, and periosteum - implications for cartilage repair

ABSTRACTPurpose: There is a clinical need to better characterize tissue sources being used for stem cell therapies. This study focuses on comparison of cells and connective tissue progenitors (CTPs) derived from native human infrapatellar fatpad (IPFP), synovium (SYN), and periosteum (PERI).Materials and Methods: IPFP, SYN, PERI were harvested from twenty-eight patients undergoing arthroplasty. CTPs were quantitatively characterized using automated colony-forming-unit assay to compare total nucleated cell concentration—[Cell], cells/mg; prevalence-(PCTP), CTPs/million nucleated cells; CTP concentration—[CTP], CTPs/mg; proliferation and differentiation potential; and correlate outcomes with patient’s age and gender.Results: [Cell] did not differ between IPFP, SYN, and PERI. PCTP was influenced by age and gender: patients >60 years, IPFP and SYN had higher PCTP than PERI (p < 0.001) and females had higher PCTP in IPFP (p < 0.001) and SYN (p = 0.001) than PERI. [CTP] was influenced by age: patients <50 years, SYN (p = 0.0165) and PERI (p < 0.001) had higher [CTP] than IPFP; patients between 60 and 69 years, SYN (p < 0.001) had higher [CTP] than PERI; patients >70 years, IPFP (p = 0.006) had higher [CTP] than PERI. In patients >60 years, proliferation potential of CTPs differed significantly (SYN>IPFP>PERI); however, differentiation potentials were comparable between all three tissue sources.Conclusion: SYN and IPFP may serve as a preferred tissue source for patients >60 years, and PERI along with SYN and IPFP may serve as a preferred tissue source for patients <60 years for cartilage repair. However, the heterogeneity among the CTPs in any given tissue source suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.

196 - The Role of Total Nucleated Cell Concentration in Bone Marrow Collections

196 - The Role of Total Nucleated Cell Concentration in Bone Marrow Collections

HLA-Identical Embryos Selected after in Vitro Fertilization and Pre-Implantation Genetic Diagnosis Combined with HLA Typing: A Promising Option for Successful Allogeneic Hematopoietic Stem Cell Transplantation for Children with Genetic or Malignant Disorders Who Lack an HLA-Matched Related or Unrelated Donor

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option for many children with various genetic or malignant diseases. However not all children in need for HSCT have a suitable donor available. For such cases in vitro fertilization (IVF) with pre-implantation genetic diagnosis (PGD) combined with human leukocyte antigen (HLA) tissue typing has been used to select an IVF healthy and HLA-matched embryo in order to give birth to a child who may serve as a stem cell donor. We report our experience with five children transplanted from HLA-matched siblings selected after IVF and PGD in our center.Methods and Patients: The clinical protocol was approved by the center’s Institutional Research and Ethics Committee. Written informed consent was obtained from parents.Hormonal stimulation for IVF, culture techniques, PGD and HLA-typing were performed according to standard protocols. Detection of the genetic disease mutation was performed by using mini-sequencing. Embryos genotyping was performed by using multiplex polymerase chain reaction (PCR) analysis of informative polymorphic short tandem repeat (STR) markers.Healthy and HLA-identical embryos were transferred 6 days after fertilization to the uterus of the respective mothers. Confirmatory genetic testing and HLA-typing were performed during the first trimester of pregnancy by using chorionic villus sampling.Patient’s characteristics are shown in Table 1. None of our patients had an available matched sibling donor. All patients underwent myeloablative conditioning consisting of busulfan, cyclophosphamide and antithymocytic globulin. GVHD prophylaxis consisted of CyA plus Methotrexate. Stem cell graft consisted of bone marrow plus cord blood in 4 out of 5 patients, while one patient received bone marrow only.Results: All couples underwent a single cycle of IVF resulting into the generation of 78 embryos. Fourteen embryos were healthy and HLA-identical with their sibling and 10 of them were transferred to the uterus of the respective mother giving birth to a total of 6 healthy children. Results of IVF, PGD and HLA-typing are shown in more detail in Table 2.The median age of the donor at the time of HSCT was 16 months. The median total nucleated cell (TNC) concentration of cord blood grafts was 2.3x107/kg, while the median CD34+ cell was 0.47x105/kg. The median TNC and CD34 cell concentration of bone marrow grafts was 1,97x108/kg and 5,09x106/kg respectively. All patients achieved sustained engraftment of donor cells. Mild acute GVHD of the skin was observed in one out of 5 patients. As of today, with a median follow up period of 4 years, all patients are alive with complete donor chimerism, without chronic GVHD and free of disease.Conclusions: IVF and PGD/HLA typing methodology should be considered and discussed with parents in cases where no HLA-matched donor is available. The procedure can be applied only in cases requiring non-urgent HSCT, and in parents of reproductive age. Table 1:Patient’s characteristicsPatientSexAge (years)DiseasePrevious treatmentsDate of transplant1Male4,5Chronic granulomatous diseaseInterferon-gamma, antibiotics2/10/20072Male4Chronic granulomatous diseaseantibiotics1/7/20083Male11Chronic myeloid leukemiaHydroxyurea, imatinib21/7/20094Male4Thalassemia majorRBC transfusions4/5/20105Female4,5Diamond-Blackfan anemiaCorticosteroids, RBC transfusions30/4/2013Table 2:Results of IVF, PGD and HLA-typingPatientsIVF (cycles)Number of embryos after IVFNumber of healthy and HLA-identical embryosNumber of healthy and HLA-identical transferred embryosNumber of healthy and HLA-identical borned children11376212111221311022141101115110332 DisclosuresNo relevant conflicts of interest to declare.

Cerebrospinal fluid eosinophilia is a sensitive and specific test for the diagnosis of Parelaphostrongylus tenuis in camelids in the northeastern United States

Aberrant migration of Parelaphostrongylus tenuis in camelids results in neurologic deficits, recumbency, and sometimes death. An antemortem diagnosis of P. tenuis in camelids is typically based upon the presence of characteristic asymmetric neurologic deficits, known exposure to white-tailed deer, cerebrospinal fluid (CSF) eosinophilia, and response to treatment. The diagnostic accuracy of CSF eosinophil percentage for the diagnosis of P. tenuis in camelids has not been critically examined. The objective of the current study was to determine the sensitivity (Se) and specificity (Sp) of CSF eosinophil percentage, CSF eosinophil concentration, total nucleated cell concentration, and protein concentration for the antemortem diagnosis of P. tenuis. Medical records of camelids admitted to Cornell University with clinical signs of neurologic disease, CSF analysis, and necropsy were examined from January 2000 through December 2009. Se and Sp were determined by receiver operating characteristic curves in camelids diagnosed with P. tenuis (n = 13) or other conditions (n = 24) based on postmortem examination. More than 17% of eosinophils in CSF had a Se of 85% and Sp of 92% for P. tenuis diagnosis (area under the curve [AUC]: 0.87; SE AUC: 0.07; P < 0.0001; 95% confidence interval [CI] AUC: 0.72-0.96), and >1.4 eosinophils/µl of CSF had a Se of 85% and Sp of 96% (AUC: 0.9; SE AUC: 0.06; P < 0.0001; 95% CI AUC: 0.76-0.97). Cerebrospinal fluid eosinophil percentage and concentration are sensitive and specific methods for diagnosing P. tenuis antemortem in camelids residing in regions endemic to white-tailed deer.

The Immunologic Composition of the Bone Marrow Changes Rapidly During the Harvest Procedure

AbstractAbstract 1915Bone marrow (BM) has been associated with a decreased rate of Graft-versus-Host-Disease (GvHD) when compared to peripheral blood stem cells (PBSC) in the context of allogeneic stem cell transplantation (allo-SCT). It has been reasoned that this may relate in part to the differential quantitative and qualitative immunologic composition of both stem cell sources. It has, for example, been noted that BM contains a higher percentage of CD8+ T-cells, natural killer (NK) cells and recently also more regulatory T-cells than PBSC. It is further known that the total nucleated cell concentration (TNC) correlates negatively with the BM harvest volume, which could argue for an increasing dilution of the transplant with peripheral blood (pB) during the BM harvest. How this impacts on the different immunological compartments and how the BM at different time points of the harvest procedure compares to PBSC products has not been determined yet.During routine BM harvest procedures (n = 24) we subjected a part of the aspirate drawn at the beginning, after withdrawing half of the prescribed volume and at the end of the harvest procedure to detailed immunophenotyping using multicolor flowcytometry including intracellular staining for FoxP3 and IL17. The PBSC products of an age matched group (n = 20) were analyzed in parallel with the same flowcytometry protocols and used as a comparison.During the BM harvest the median TNC dropped rapidly from 53.2 Gpt/l in the beginning to 17.0 Gpt/l after half the volume had been collected and finally reached 6.2 Gpt/l at the end of the procedure. As expected, the first BM specimen contained a higher concentration of NK- (6.7 vs. 4.6 %, p = 0.001) and B-cells (14.1 vs. 10.2 %, p < 0.001) but less T-helper-(Th)-cells (36.0 vs. 53.7 %, p < 0.001) than pB. After collection half of the prescribed volume the NK-cell concentration had already dropped to 4.5 %, which was not significantly different from pB (p = 0.412) anymore. The B-cell concentration, however, remained at levels comparable to the first specimen (14.5 %) and significantly differed from pB (p < 0.001). The last BM specimen drawn still had a higher B-cell concentration than pB (10.9 vs. 10.2 %), although this was not statistically significant (p = 0.265). Th-cells demonstrated a steady increase in concentration during the harvest with a median concentration of 43.4 and 47.0 % in the middle and at the end of the procedure. Both the halfway value and the end of harvest concentration were significantly different from pB (p < 0.001 and p = 0.004, respectively). An overview on the NK-, B- and Th-cell-concentrations at the different time points analyzed can be found in Figure 1.Within the Th compartment we were unable to see a clear trend for Treg which had a concentration of 7.6 % in the beginning, 7.0% halfway and 7.1 % after all BM had been collected. None of these concentrations was significantly different from that found in pB (7.6 %). The same applied to Th17 cells which made up 0.51 % of Th in the beginning, 0.59 % in the middle and 0.50 % at the end of the procedure and 0.84 % in the pB. Moreover there was no significant difference between the first BM specimen and PBSC products with respect to Treg (7.6 vs. 6.7 %, p = 0.181) or Th17 (0.51 vs. 0.61 %, p = 0.100). In contrast, the NK- and Th-cell concentration in PBSC products was significantly higher than in the first BM specimen (NK: 10.7 % vs. 6.7 %, p =0.005; Th: 40.3 % vs. 36.0 %, p =0.038).We conclude that the composition of immune cells within the BM changes significantly during the harvest procedure probably due to an increasing dilution with peripheral blood. These changes variably affect different compartments and may have an impact on post-transplant immunological function and complications. Therefore BM harvest volumes may need to be considered when comparing BM and PBSC with respect to clinical outcomes. In contrast to previous reports, we found no indication that BM, no matter at which time point analyzed, contained a higher concentration of Treg when compared to PBSC. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

How do I collect and process stem cells?

How do I collect and process stem cells?

Prediction of nucleated cells in bone marrow stem cell products by donor characteristics: a retrospective single centre analysis

Number of total nucleated cells is an important prognostic factor of unrelated bone marrow transplantation. The objective of our study was to further optimize donor selection by finding donor characteristics which predict the total nucleated cell concentration in unrelated bone marrow products. Data were collected retrospectively from the pre-donation medical records. The outcome variable was concentration of total nucleated cells in the final products. We investigated several candidate predictors, including demographic variables and peripheral blood counts. A backward selection procedure was performed, using multivariable multilevel analysis. A total of 471 donations were included. Significant predictors were increase of age (ratio per 5 years 0.971), cytomegalovirus (ratio 0.947 if positive), smoking (ratio 1.079 if yes), higher haemoglobin (ratio 1.052 per mmol/l), higher mono-nuclear cells (ratio 1.169 when mono-nuclear cells >2.69 compared to mono-nuclear cells <1.89 x 10E9/l), increasing number of whole blood donations (ratio 1.115 when blood donations >4 compared to blood donation <1), and larger body surface area (ratio 1.179 when body surface area >2.07 compared to body surface area <1.82 m(2)). The model was adjusted for collection volume. Presence of all favourable factors was associated with a twofold higher concentration of total nucleated cells in the bone marrow harvest.

Cytologic interpretation of canine cerebrospinal fluid samples with low total nucleated cell concentration, with and without blood contamination

Cerebrospinal fluid (CSF) is potentially altered by iatrogenic blood contamination at the time of sampling due to the addition of blood-associated leukocytes and protein. The objective of this study was to assess whether protein concentration, neutrophil percentage, and the presence of activated macrophages, reactive lymphocytes, or eosinophils in CSF samples with low total nucleated cell concentration (TNCC) are affected by blood contamination or associated with central nervous system (CNS) disease. Case records from the Royal Veterinary College Diagnostic Laboratory were searched retrospectively for dogs with CSF having < or =5 TNCC/microL. TNCC, RBC, and protein concentrations; neutrophil percentage; and the presence of activated macrophages, reactive lymphocytes, and eosinophils were recorded. Results of magnetic resonance imaging (MRI) also were recorded as a marker of CNS disease. Of 906 cases evaluated, 106 (12%) had blood contamination (>500 RBCs/microL) in CSF. Protein concentration and neutrophil percentage were significantly higher and the presence of eosinophils was more likely in blood contaminated vs noncontaminated samples. Non-blood-contaminated samples with activated macrophages or reactive lymphocytes had higher protein concentrations and neutrophil percentages, and those with activated macrophages were more likely to have a positive finding on MRI. Protein concentration, neutrophil percentage, and the presence of eosinophils are significantly affected by blood contamination in canine CSF having low TNCC. Activated macrophages and reactive lymphocytes are not affected by blood contamination, however, and may be useful in identifying dogs with CNS abnormalities.

What is your diagnosis? Muculent pleural effusion from a dog

Pleural effusion was examined from a 5-year-old, female Brittany Spaniel with a 7-day history of dyspnea, anorexia, and diarrhea. The fluid was yellow, cloudy, and slightly gelatinous, and had a total protein concentration of 2.8 g/dL, a total nucleated cell concentration of 1.1 x 10(3)/microL, and a triglyceride concentration of 177 mg/dL. A cytocentrifuged preparation contained a mixed inflammatory cell population with a predominance of small lymphocytes and abundant mucinous material in the background. The dog died 3 days later and a mass was found within the lumen and wall of the right auricle of the heart at necropsy. Histopathologic sections of the mass contained a population of anaplastic spindle cells diffusely suspended in a pale basophilic matrix, consistent with myxosarcoma. The cells were positive for vimentin and negative for cytokeratin, desmin, and von Willebrand factor VIII-related antigen. A myxoid matrix was confirmed by positive staining with Alcian blue. Myxosarcoma is a rare cardiac tumor in dogs that should be considered, along with mucus-producing carcinomas and bile, as a cause of muculent effusion.