Total Nucleated Cell Count Research Articles

Diagnostic utility of the total nucleated cell count for differentiation of septic and sterile peritoneal effusions in dogs.

Rapid and accurate diagnosis of septic peritonitis is critical for initiating appropriate medical and surgical management. The aim of this study was to determine the diagnostic utility of the total nucleated cell count (TNCC), absolute neutrophil count, neutrophil percentage, and total protein (TP) to distinguish septic versus non-septic peritoneal effusions in dogs. Electronic medical records were retrospectively searched for peritoneal fluid samples from 2008 to 2018 and classified as septic or non-septic based on bacterial culture and/or cytology results. Receiver operator characteristic curves (ROCs) were used to describe the overall diagnostic utility of each test, with optimal cutpoints analyzed to dichotomize continuous variables. Positive and negative likelihood ratios were calculated at these cutpoints. A total of 166 unique samples, including 87 septic and 79 non-septic peritoneal effusions, were included. There were no significant differences in dog sex, age, or days hospitalized between groups. Septic effusions had significantly higher TP, TNCC, absolute neutrophil count, and neutrophil percentage compared with non-septic effusions. The area under the curve of the ROC curves was TNCC (0.80), absolute neutrophil count (0.80), neutrophil percentage (0.64), and TP (0.63). For TNCC and absolute neutrophil count, optimal cutoffs were 17.13 × 103 cells/μL and 19.88 × 103 cells/μL, resulting in positive and negative likelihood ratios of 2.39 and 0.28 and 2.85 and 0.28, respectively. Total nucleated cell counts and absolute neutrophil counts aid in the differentiation of septic and non-septic peritoneal effusions with similar diagnostic utility but are not sufficiently sensitive or specific to use without concurrent microscopic evaluation.

Comparison of Sysmex XN-V body fluid mode and deep-learning-based quantification with manual techniques for total nucleated cell count and differential count for equine bronchoalveolar lavage samples.

Bronchoalveolar lavage (BAL) is a diagnostic method for the assessment of the lower respiratory airway health status in horses. Differential cell count and sometimes also total nucleated cell count (TNCC) are routinely measured by time-consuming manual methods, while faster automated methods exist. The aims of this study were to compare: 1) the Sysmex XN-V body fluid (BF) mode with the manual techniques for TNCC and two-part differential into mononuclear and polymorphonuclear cells; 2) the Olympus VS200 slide scanner and software generated deep-learning-based algorithm with manual techniques for four-part differential cell count into alveolar macrophages, lymphocytes, neutrophils, and mast cells. The methods were compared in 69 clinical BAL samples. Incorrect gating by the Sysmex BF mode was observed on many scattergrams, therefore all samples were reanalyzed with manually set gates. For the TNCC, a proportional and systematic bias with a correlation of r = 0.79 was seen when comparing the Sysmex BF mode with manual methods. For the two-part differential count, a mild constant and proportional bias and a very small mean difference with moderate limits of agreement with a correlation of r = 0.84 and 0.83 were seen when comparing the Sysmex BF mode with manual methods. The Sysmex BF mode classified significantly more samples as abnormal based on the TNCC and the two-part differential compared to the manual method. When comparing the Olympus VS200 deep-learning-based algorithm with manual methods for the four-part differential cell count, a very small bias in the regression analysis and a very small mean difference in the difference plot, as well as a correlation of r = 0.85 to 0.92 were observed for all four cell categories. The Olympus VS200 deep-learning-based algorithm also showed better precision than manual methods for the four-part differential cell count, especially with an increasing number of analyzed cells. The Sysmex XN-V BF mode can be used for TNCC and two-part differential count measurements after reanalyzing the samples with manually set gates. The Olympus VS200 deep-learning-based algorithm correlates well with the manual methods, while showing better precision and can be used for a four-part differential cell count.

Expressão de TLR-2, FOXP3 e COX-2 na membrana sinovial de cães com artrite induzida pela leishmaniose canina

ABSTRACT: Canine leishmaniasis (CanL) is a multifaceted disease triggered by the protozoan Leishmania infantum, characterized by diverse clinical presentations, including osteoarticular complications. Immune-mediated joint diseases invariably initiate at the synovial membrane, implicating its pivotal role in arthritis pathogenesis. This study aimed to investigate the influence of natural L. infantum infection on synovial fluid characteristics and the expression of immune markers, including TLR-2, FOXP3, and COX-2, in the synovial membrane. Twenty naturally infected dogs (NID) with L. infantum were sourced from the Zoonosis Surveillance Unit (ZSU). Clinical-orthopedic assessments were conducted, encompassing lameness, joint edema, crepitus, patellar luxation, and the drawer test. Synovial fluid (SF) parameters, including volume, appearance, viscosity, total nucleated cell count (TNC), neutrophil count, and total protein (TP) content, were determined. After anesthesia and euthanasia, synovial membrane specimens were obtained. SF protein concentrations categorized dogs into three groups: GI (2 to 2.5g/dL), GII (2.5 to 6.0g/dL), and GIII (>6g/dL). Inflammatory infiltrates and synovial membrane changes were assessed, and immunohistochemistry evaluated TLR-2, FOXP3, and COX-2 marker expressions. Clinical evaluations revealed various osteoarticular abnormalities in NID dogs, including lameness (55%), joint edema (25%), crepitus (30%), patellar luxation (20%), and positive drawer test (25%). Post mortem examinations revealed bilateral subchondral bone, meniscus, and trochlea erosion in 30% of cases. Amastigotes of L. infantum were identified extracellularly and within macrophages (60%). An inflammatory infiltrate was predominant in 70% of dogs, with varying intensity among the groups. Mononuclear cells, chiefly macrophages and lymphocytes, and neutrophils comprised the infiltrate. TLR-2 and COX-2 expression levels were elevated in GIII compared to GII and GI. Conversely, FOXP3 showed moderate expression in GI and minimal expression in GII and GIII. This study underscores the contributory role of L. infantum infection in the development of joint lesions in CanL. Additionally, alterations in the expression of immune markers TLR2, FOXP3, and COX2 within the synovial membrane imply the perpetuation and exacerbation of the inflammatory processes, shedding light on the intricate pathogenesis of CanL-induced arthritis.

Single CBT As 2nd Allo-SCT Is Promising for Selected Patients with Relapsed Hematopoietic Malignancies

Background: Hematopoietic malignancies that relapse after allogeneic stem cell transplantation (allo-SCT) generally have a poor prognosis, and there is no standard treatment. Second allo-SCT is a potentially curative treatment, but many patients do not receive 2nd allo-SCT at the best time because of the long preparation time for transplantation, and even if they do, treatment-related complications and relapse of the underlying disease often lead them to fatal outcomes. Umbilical cord blood is considered to be a promising source of 2nd allo-SCT because of its short preparation time, rapid and high graft-versus Leukemia (GVL) effect after transplantation, and better response to GVHD treatment compared to other donor sources. Therefore, we investigated the usefulness of cord blood transplantation for relapsed hematopoietic malignancies after allo-SCT. Methods: We retrospectively analyzed the outcomes of patients with hematopoietic malignancies who received single cord blood transplantation (CBT) as 2nd allo-SCT for relapse after allo-SCT in Toranomon Hospital between Apr. 1999 and Mar. 2022. We exclude the patients with Eastern Cooperative Oncology Group (ECOG) performance status(PS) of 4. Results: 263 patients were included in this study. There were 179 patients with myeloid neoplasms and 84 with lymphoid neoplasms. Median age was 45 years (range: 18-71). 239 patients were in non-remission and 24 in remission before SCT. Median interval between 1st SCT and 2nd SCT was 293 days (26-4703). Donor sources of 1st allo-SCT were related donor in 80, unrelated donor in 62, and sCBT in 121 patients. 135 patients received myeloablative conditioning for 2nd allo-SCT, and 128 patients underwent reduced intensity-conditioning. About cord blood as the donor source of 2nd allo-SCT, the median total nucleated cell count at cryopreservation was 2.71(×10 7)/kg (1.67-5.85), and median CD34 + cell count at cryopreservation was 0.97(×10 5)/kg (0.25-3.18). Median observation period of survivors was 1726 (122-6286) days after 2nd allo-SCT. Overall survival (OS), non-relapse mortality and relapse at 2-year were 16.7%, 46.9% and 40.6%, respectively. Younger patients (< 50 years) had significantly better OS than older patients (50 years and more), with 2-year OS of 21.5% and 9.9%, respectively (P < 0.001). Patients with good PS (0 and 1) had significantly better survival than those with poor PS(2 and 3), with 2-year OS of 23.4% and 2.4%, respectively (P<0.001). Patients with myeloid malignancies tended to have better survival than those with lymphoid malignancies, with 2-year survival rates of 19.2% and 11.9%, respectively (P=0.341). Patients with longer interval between SCTs (>1 year) had significantly better survival than those with shorter interval (<1 year), with 2-year OS of 23.6% and 11.6%, respectively (P=0.00443). In multivariate analysis, younger age (<50), good PS (0-1), long interval between SCTs(≧1 year) showed a superior OS. In the younger age population, patients with myeloid neoplasms showed a superior OS compared to those with lymphoid neoplasms (2yrOS: 30.3% vs 10.6%, P=0.00318). Furthermore, patients with myeloid neoplasms having good PS and long interval between SCTs showed an extremely high survival rate of 50.5% at 2 years (n=31)(Figure 1). In the elderly population, patients with long interval and good PS showed a relatively good OS of 31.4% at 2 years (n=28)(Figure 2). The type of disease (myeloid vs lymphoid) didn't have a significant influence on OS in this subpopulation (28.1% vs 50.0%, P=0.129). Conclusion: Although the outcomes of CBT as 2nd allo-SCT haven't been satisfactory, there are certain populations, including elderly patients, with favorable OS.

Physicochemical and Cytological Analysis of Synovial Fluid in Young and Adult Dogs

Background: Synovial fluid is a complex viscous fluid which provides lubrication to the articulating joints to avoid friction and wearing of cartilages. It is also an indicator of health status of joint, but a comparison of its composition between young and adult dogs is lacking. Methods: Present study was conducted to evaluate physical, biochemical and cytological analysis of synovial fluid in apparently healthy stifle joint of young and adult non-descript dogs. Twelve non-descript dogs irrespective of sex were divided into two groups (6 dogs in each group) i.e., group I (2-8 months of age) and group II (1-8 years of age). Dogs died in the OPDs of Veterinary Clinical Complex, Jabalpur were acquired with the consent of pet parent. The duration of study was one year, i.e., from September 2021 to August 2022. Immediately after collection of synovial fluid; volume, colour, viscosity, pH, specific gravity and refractive index were recorded, smear on slides were prepared. Protein and glucose were estimated within one hour using biochemical semi-autoanalyzer. Result: Volume of fluid collected in a single attempt varied from one drop to 0.5 ml. Mean pH values in group I and II were 7.33±0.17 and 7.67±0.25 respectively. Mean values of refractive index and specific gravity were 1.34 and 1.02 respectively in both the groups. Observations of mucin clot test of synovial fluid in group I and II exhibited a tight ropy clot in a clear solution. Turbidity was not observed in any case. Mean protein concentration was 1.29±0.18 g/dl and 2.74±0.84 g/dl in group I and II respectively. The concentration of glucose in group I and II was 102.79±3.66 mg/dl and 105.47±4.19 mg/dl respectively, that was around normal blood plasma level. Cytological observation revealed 1-3 cells per field at 1000x magnification. Mean neutrophil count in both the groups was about 6% of total nucleated cell count. Small mononuclear cells and large mononuclear cells were 19±3.79, 74.33±4.24 and 12.17±2.33, 81.83±2.54 in group I and II respectively. Biochemically and cytologically no significant difference was observed between the group I and II. Vacuolated large mononuclear cells contributed about 6-6.5% of total large mononuclear cells in both the groups. No significant difference was observed in most physical and biochemical parameters of synovial fluid in young and adult dogs, except a higher concentration of protein content in adults.

CHARACTERIZING CELLULAR COMPONENTS OF BONE MARROW ASPIRATE CONCENTRATE: A ONE-STEP CELL THERAPY FOR ANKLE CARTILAGE DEFECTS

AbstractObjectivesBone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration.MethodsDuring surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec®, UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD.ResultsMean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively).ConclusionsBMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes.Declaration of Interest(a) fully declare any financial or other potential conflict of interest

NCOG-28. RECURRENCE OF PLEOMORPHIC XANTHOASTROCYTOMA PRESENTING WITH DIFFUSE LEPTOMENINGEAL SPREAD

Abstract Pleomorphic xanthoastrocytomas (PXAs) are rare primary brain tumors that mainly affect children and young adults. We report a case of recurrent PXA in an adult and review the literature. To our knowledge, this is the first adult report highlighting clinical, CSF, and imaging findings of recurrent PXA with leptomeningeal spread. A 60-year-old woman presented with a 2-month history of fluctuating mental status, aphasia, low-back pain, progressive paraparesis, and urinary retention. She was previously diagnosed with a left temporal PXA 17 years prior and underwent gross total resection with no evidence of tumor recurrence for the next several years. Approximately 10 months prior to her current presentation, she was found to have radiographic recurrence of the left temporal PXA. She underwent gross total resection, with final pathology revealing anaplastic transformation, WHO grade 3, BRAF V600E mutation positive. She next underwent photon radiotherapy, and chemotherapy with temozolomide. She had good clinical and radiographic response to treatment for 8 months prior to developing her current symptomatology. MRI of the brain and spine showed local recurrence with new diffuse contrast enhancement of the spinal cord and nerve roots. Cerebrospinal fluid (CSF) analysis showed a total nucleated cell count of 10/mcL, protein >1250 mg/dL, glucose < 20 mg/dL, and xanthochromia. CSF cytology was positive for malignant cells consistent with PXA. Although PXA is often associated with a favorable prognosis, recurrence with anaplastic transformation can be associated with dissemination throughout the nervous system and worse outcomes. Despite the rarity of such cases, clinicians should remain vigilant, especially when anaplastic transformation occurs. Early detection of leptomeningeal involvement through thorough CSF analysis and appropriate imaging can facilitate prompt intervention and potentially improve patient outcomes.

Hematological Parameters: Comparative Evaluation of Performance between Dxh 500 and XN-10 Equipments

Introduction The development of hematopoietic stem cell transplantation (HSCT) was an extremely important milestone for treating certain malignant and non-malignant hematological diseases. The HSCT process contemplates collecting hematopoietic progenitor cells (HPC) from the donor, conditioning chemotherapy, infusion of HPC, and engraftment. Mobilized peripheral blood is the most commonly used HPC source for HSCT due to its ease of obtaining through an apheresis procedure and high progenitor cell count yields compared to bone marrow source, which requires a bone marrow aspiration procedure, usually performed in a surgical center under general anesthesia, and requires the collection of higher volumes to reach the amount of HPC desired. The total nucleated cell count (TNC) corresponds to the sum of all nucleated cells in the sample and is used as a reference for the target dose for both sources of HPC. The TNC provides essential information on the quality of the sample obtained, since an infusion of insufficient number of cells affects the ability to reconstitute the recipient's hematopoietic system. An insufficient TNC can result in poor engraftment, leading to a slow recovery of blood cell production and increasing the risk of complications such as infections and bleeding. Determination of the number of nucleated cells in bone marrow and peripheral blood samples can be performed automatically by hematological analyzers such as the Beckman Coulter DxH 500 and Sysmex XN-10. This study aims to compare the performance of those equipments in determining hematological parameters in bone marrow and apheresis material. Materials and methods We compared the results of 40 HPC samples (20 bone marrow samples and 20 peripheral blood samples collected by apheresis) analyzed in the Sysmex XN-10 and Beckman Coulter DxH 500 equipments. All these specimens were analyzed without dilution and diluted 1:10. The evaluated parameters were: TNC, hematocrit, platelets, lymphocytes, monocytes, and granulocytes. Results The analysis of the results showed a positive Pearson correlation coefficient (above 0.9) between XN-10 and DxH 500 equipment for the evaluation of TNC in diluted samples obtained by apheresis (figure 1) and by bone marrow (figure 2). In pure samples obtained by apheresis the DxH 500 equipment showed non-linearity for TNC but revealed a positive correlation with the XN-10 when evaluating pure bone marrow samples (figure 2). On the other hand, XN-10 equipment showed linearity when correlating TCN between pure and diluted samples obtained by apheresis (figure 1) and bone marrow aspiration (figure 2). Hematocrit analysis in samples obtained by apheresis showed 90% and 100% of agreement for diluted samples between the equipment and for diluted versus pure samples, respectively, when using the cutoff value of 12%. Hematocrit validation using the Beckman Coulter equipment was impaired due to the sample's viscosity and limited linearity. Also, there was a satisfactory hematocrit correlation for pure bone marrow samples and comparing diluted versus pure bone marrow samples with both equipment. The results also showed a good correlation when analyzing the platelet count in pure and diluted samples (bone marrow and apheresis) with XN-10 or DxH 500, as well as comparing the diluted samples versus the pure samples. The use of differential count of nucleated cells (lymphocytes, monocytes and granulocytes) realized by the two evaluated devices was not approved for bone marrow or apheresis samples. Conclusion The determination of TNC, platelet and hematocrit counts using the Sysmex XN-10 equipment were satisfactory for both pure and diluted samples obtained by apheresis or bone marrow. For pure samples obtained by apheresis analyzed by DxH 500, we could observe absence of linearity for TNC and hematocrit. This situation could be explained by the high number of cells in this sample and the consequent high viscosity.

Adaptive Manufacturing of LV20.19 CAR T-Cells for Relapsed, Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy characterized by the presence of t(11;14) and bright CD20 expression. Approved CD19 CAR T-cell therapy for relapsed/refractory (R/R) MCL is limited by both relapse and high rates of Grade 3+ cytokine release syndrome (CRS) and ICANS with 15% and 31% patients (pts) experiencing such toxicities, respectively. To improve outcomes we utilized dual targeted lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T-cells with an adaptive manufacturing (MF) process to optimize the final CAR product as part of a Phase 1/2 clinical trial in R/R MCL. Methods: We conducted a Phase 1/2 single center, prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R B-cell non-Hodgkin Lymphoma. We report results of MCL pts from a fully enrolled Phase I safety run-in and Phase II efficacy cohort. The primary outcome for the Phase II cohort was day 90 complete response (CR) rate. The Phase II cohort utilized an adaptive 8/12 day MF process to enhance the percentages of T-SCM (stem cell-like memory) and T-CM (central memory) CAR T-cells in the final product by shortening culture time. CAR T-cells were harvested either on day 8 of MF or extended to a 12-day MF process depending on achieving target cell dose in culture. Fludarabine/cyclophosphamide lymphodepletion was started during MF to facilitate fresh infusion in eligible pts. Descriptive statistics were utilized for demographic data and the Kaplan Meier methodology for survival analysis. Results: In total 17 pts with R/R MCL received LV20.19 CAR T-cells (Phase 1=3 pts and Phase 2=14 patients). All pts achieved target cell dose. 13 pts received an 8-day product while 4 received a 12-day product. 14/17 pts received a fresh (non-cryopreserved) infusion. 8-day products were enriched for T-SCM/CM phenotype cells with significantly lower number of terminally differentiated effector memory (EMRA) T-cells in the CD4 compartment (p<0.05) than 12-day products. The median age was 63 (50-74 years) and 15 (88%) were cisgender male. The median number of lines of therapy was 4 (3-8). p53 deletions or mutation were present in 7 pts. Day 28 overall response rate (ORR) was 100%; CR rate=76% and partial response (PR) =24%. Initial ClonoSEQ MRD assay performed within the first 90 days of LV20.19 CAR T (n=13) was negative in 10 pts (77%). Among Phase II patients with day 90 results available (n=12) the ORR was 100%, CR rate=92% and PR rate=8% exceeding the Phase II efficacy threshold. Two pts have relapsed to date, +8 months and +2 years after infusion. There were two non-relapse mortality (NRM) events that occurred beyond day 28: gram negative rod sepsis & COVID19 infection resulting in a 1-year NRM rate of 12%. The 1-year progression free survival (PFS) was 77%, 1-year duration of response (DOR) was 92%, and 1-year overall survival (OS) of 84%. Median PFS/DOR/OS have not been met with median follow-up of 14 months of surviving pts (Figure 1). In terms of safety, 94% (n=16) experienced CRS, all grade 1-2. 18% (n=3) had ICANS in the first 28-days, 2 pts with Grade 3 toxicity. There were two pts with late (>day 28) episodes of Grade 1 neurological toxicity. Both pts had immense expansion of lymphocytes in the CSF (total nucleated cell count of 386/µLand 1005/µL) with CAR+ T cells present. Both received only a single dose of intrathecal hydrocortisone (no systemic steroids) with resolution of neurotoxicity within 24 hours of administration. Conclusions: Bispecific LV20.19 CAR T-cells with adaptive MF process is feasible, safe, and efficacious for R/R MCL with ORR 100%, no Grade 3+ CRS and low rates of Grade 3+ ICANS (12%). Adaptive MF enriched the final product with higher percentages of T-SCM/T-CM CAR cells and allowed most pts to receive CAR-T cells within 8 days of apheresis. Dual targeting of CD20 and CD19 with CAR-T cells may improve outcomes in pts with relapsed, refractory MCL.

Evaluation of a Manual Cytocentrifuge versus the Standard Automated Cytocentrifuge in the Analysis of Canine Cerebrospinal Fluid: A Case Series of 55 Dogs.

Cytospins are important for evaluating fluids with very low cellularity such as cerebrospinal fluid (CSF). The aim of this study was to compare the CSF cytospin preparations obtained from automated and manual cytocentrifugation methods. A prospective case series was performed to analyze canine CSF samples using both centrifugation methods. The cytospins were processed within 30-60 min and prepared simultaneously in a conventional automated cytocentrifuge and in an in-house manual cytocentrifuge, using a fixed volume of CSF fluid. The cellularity, differential cell count and the proportion of cell artifacts (pseudopods and vacuolization) were blindly assessed in the cytospin preparations obtained using the two methods. The agreement and correlation between both methods were analyzed. There were 55 dogs enrolled (48 prospectively and 7 retrospectively) in the study. 38 dogs had normal total nucleated cell counts, while 17 had pleocytosis. Automated and manual cytocentrifugation had similar cell yields, and no significant differences in differential cell counts or the presence of artifacts existed between both methods. In cases with pleocytosis, the cytologic diagnosis obtained using each method was similar. Manual cytocentrifugation of CSF is a reliable and economic method designed for routine clinical practice. Its use reduces the specimen deterioration related to processing and analysis delays when samples are transported to external laboratories for evaluation.

Intra-articular bone marrow mononuclear cell therapy improves lameness from naturally occurring equine osteoarthritis.

Osteoarthritis (OA) can be debilitating and is related to impaired resolution of synovial inflammation. Current treatments offer temporary relief of clinical signs, but have potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that have the ability to reduce OA symptoms in people and inflammation in experimentally-induced synovitis in horses. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatment groups: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Lameness was evaluated subjectively and objectively, joint circumference measured, and synovial fluid collected for cytology and growth factor/cytokine quantification at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at p < 0.05. There were no adverse effects noted in any treatment group. There was a significant increase in synovial fluid total nucleated cell count in the BMNC-treated group on day 7 (median 440; range 20-1920 cells/uL) compared to day 0. Mononuclear cells were the predominant cell type across treatments at all time points. Joint circumference decreased significantly in the BMNC-treated group from days 7 to 21 and was significantly lower at day 21 in the BMNC-treated group compared to the saline-treated group. Median objective lameness improved significantly in the BMNC group between days 7 and 21. GM-CSF, IL-1ra, IGF-1, and TNF-α were below detectable limits and IL-6, IL-1β, FGF-2 were detectable in a limited number of synovial fluid samples. Inconsistent and limited differences were detected over time and between treatment groups for synovial fluid PGE2, SDF-1, MCP-1 and IL-10. Decreased lameness and joint circumference, coupled with a lack of adverse effects following BMNC treatment, support a larger clinical trial using BMNC therapy to treat OA in horses.

Abstract 9 Identification of Effective Changes to Workflow Triaging Criteria and Production Planning

Abstract Introduction The Cleveland Cord Blood Center (CCBC) analyzes fresh incoming cord blood units for the total nucleated cell (TNC) count. Units that meet the TNC criteria are processed for clinical banking and units not meeting the defined criteria are made available for research collaborations. CCBC sought to understand if adding additional pre-screening criteria would reduce post-manufacturing deferral rates and increase production of licensed units. Objectives To identify the main failure modes resulting in cord blood unit deferral for clinical banking post-manufacturing. To identify key factors resulting in unlicensed status of processed cord blood units. To implement changes to pre-screening strategies to reduce deferral rates and increase production of licensed units. Methods CCBC created four cross-departmental teams to evaluate the main reasons for deferrals post-manufacturing and to evaluate travel-risk history. Each committee had representation from collections, quality, and laboratory staff. Data was evaluated that was collected between January and June 2022. Results The committees identified that 19% of all deferrals post-manufacturing were related to low CD34 count and/or viability and that 17% of all processed units were unlicensed due to travel to Zika related areas. In March 2023, pre-screening criteria was updated to include a pre-processed CD34 count and viability and a pre-screening of the donating mother for travel history after consenting and prior to manufacturing. Units that fulfilled both the TNC and CD34 cut offs and did not have a high-risk travel history were processed for clinical banking. Data evaluated from March and April 2023 indicated that the deferral rates for low viable CD34 counts post-manufacturing went from 19% down to 0% and the percentage of high-risk travel units manufactured was reduced from 17% to 7%. Discussion Review of failure modes identified two main factors to guide improved triage efficiency. By implementing a pre-processing CD34 count, the larger TNC and CD34 cord blood units were identified for banking for clinical use. By using a pre-screening process at collection sites, high-risk travel donations were identified prior to processing. In the two-month timeframe evaluated, the overall deferral rates were reduced by roughly 7% and post-manufacturing licensure status increased from 83% to 93%.

Septic inflammation of the bicipital bursa: clinical, imaging, and surgical findings in nine horses.

To describe the etiologies, clinicopathologic findings, diagnostic modalities employed, treatments, and outcome associated with cases of septic bicipital bursitis. 9 horses. Medical records of horses diagnosed with septic bicipital bursitis between 2000 and 2021 were reviewed. Horses were included if synoviocentesis of the bicipital bursa revealed a total nucleated cell count of ≥ 20,000 cells/µL with a neutrophil proportion of ≥ 80%, a total protein concentration of ≥ 4.0 g/dL, and/or the presence of bacteria on cytology, or positive culture of the synovial fluid. Information retrieved from medical records included signalment, history, clinicopathologic variables, diagnostic imaging findings, treatment, and outcome. Trauma was the most common inciting cause (n = 6). Synoviocentesis using ultrasonographic guidance was performed in all cases and showed alterations consistent with septic synovitis. Radiography identified pathology in 5 horses, whereas ultrasonography identified pathology in all horses. Treatment consisted of bursoscopy (n = 6) of the bicipital bursa of which 1 was performed under standing sedation, through-and-through needle lavage (3), bursotomy (2), or medical management alone (2). Five (55.6%) horses survived to discharge. Long-term follow-up was available for 3 horses and all were serviceably sound, with 2 in training as pleasure horses and 1 case continuing retirement. Ultrasonography was the most informative imaging modality and paramount in obtaining synovial fluid samples for definitive diagnosis of septic bicipital bursitis. Bursoscopy performed under standing sedation is a feasible treatment option. Horses treated for bicipital septic bursitis have a fair prognosis for survival and may return to some level of athletic performance.

Effect of Lavage Solution Type on Bronchoalveolar Lavage Fluid Cytology in Clinically Healthy Horses.

Equine bronchoalveolar lavage (BAL) is usually performed with 250-500 mL of isotonic saline at pH 5.5. The acidic pH of saline may cause an increase in airway neutrophil count 48 h after BAL. Other isotonic solutions such as Ringer's solution, phosphate-buffered saline (PBS) or Plasma-Lyte 148® have a neutral pH of 7.4 and might be a better choice for BAL by not provoking inflammation and the influx of neutrophils into airways. BAL was performed in four healthy horses in four different lung lobes using four different solutions in a randomized crossover design. In each lobe, BAL was performed twice with a 48 h interval using 250 mL of solution. Automated total nucleated cell counts (TNCs) were recorded, and differential cell counts in lavage fluid were determined by two investigators blinded to treatments. The mean volume of BAL fluid retrieved was 51 ± 14%. The mean neutrophil percentage (%N) increased from 1.5 ± 0.9% to 14.7 ± 9.6% at 48 h (p < 0.001) but was not significantly affected by the solution used or the lung lobe sampled. In conclusion, in this study, the influx of neutrophils into airways after BAL was independent of the type of isotonic solution used and the lung lobe sampled. Saline remains an appropriate solution for BAL in horses.

Lymphocyte immunophenotyping and concentration of MMP-9 in transudates and exudates in horses

This study is the first to provide information on the lymphocyte subpopulations in peritoneal effusions in horses. Peritoneal transudates (n = 12), peritoneal exudates (n = 6) and a pleural exudate (n = 1) were analyzed. The total nucleated cell count (TNCC), total protein (TP) and matrix metalloproteinase-9 (MMP-9) concentration determined by ELISA were measured and routine cytological evaluation was performed. CD3, CD4, CD8 and CD21 positive cells were detected by flow cytometry. A higher percentage of neutrophils (P < 0.05) and higher MMP-9 (P < 0.01) levels were found in exudates. A higher percentage of macrophages (P < 0.05) and lymphocytes (P < 0.01) were found in transudates. CD4 + lymphocytes were the most common lymphocyte subpopulation in all samples. CD21 + lymphocytes were the least common in all samples. A large variability in the percentage of CD21 + lymphocytes was found in exudates. The percentage of CD21 + lymphocytes positively correlated with the level of total protein (r = 0.5704, P < 0.05). The correlation was even stronger in the group of exudates. The percentages of lymphocyte subpopulations did not correlate with the level of MMP-9 or with cytological findings. The level of MMP-9 positively correlated with the percentage of neutrophils (r = 0.4980, P < 0.05), the level of TP (r = 0.7855, P < 0.01) and TNCC (r = 0.6129, P < 0.01). A significantly higher level of MMP-9 was detected in euthanized horses than in horses that survived (P < 0.05). However, it was shown that the level of MMP-9 in the peritoneal fluid can change significantly in a short time. More studies on repeated abdominocentesis could contribute to elucidating the role of MMP-9 as a prognostic indicator.

Stem cell mobilizating effect of heparin in patients undergoing autologous stem cell transplantation.

Adequate stem cell collection is essential for successful stem cell transplantation. Heparin enhances stem cell mobilization by competing with heparin sulfate proteoglycans. Heparin is also used as an anticoagulant before leukapheresis. Here, we evaluated the effects of heparin on stem cell mobilization in patients who underwent autologous stem cell transplantation (ASCT). We evaluated patients who underwent ASCT. Patients were divided into two groups: those who received heparin plus citrate (heparinized patients) and those who received citrate only (nonheparinized patients) for anticoagulation. Univariate and multivariate analyses were also performed. The collection efficiency 2 (CE2) for CD34+ cells was calculated and compared between heparinized and nonheparinized patients. This study included 1017 patients. There were 478 (47%) heparinized and 539 (53%) nonheparinized patients. The number of collected CD34+ cells was significantly higher in heparinized patients (P < .00001). The multivariate analyses showed that using heparin was an independent positive factor for collected CD34+ cells (adj-R2 = 0.744; F = 369.331, P < .00001). CE2 was significantly higher in heparinized patients than in nonheparinized patients (66.8% vs 52.1%; P < .00001). The rate of collecting at least 2 × 106 /kg CD34+ cells was 3.3 times higher for heparinized patients in poor mobilizers (P < .00001). Heparinized patients had significantly higher total nucleated and mononuclear cell counts (P < .00001 and <.00001, respectively). Heparin enhances stem cell collection and increases CE2. The use of heparin may reduce the need for other strategies to increase stem cell mobilization.

Effect of dipotassium-ethylenediaminetetraacetic acid or lithium-heparin treatments and storage times on selected clinicopathologic analytes in equine synovial fluid.

Sample processing methods and storage time affect the outcome of biochemical analysis. We aimed to assess the effects of dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) and lithium-heparin treatments and storage times on selected analytes in equine synovial fluid (SF). Approximately 2 mL of SF from each horse (n = 7) were collected via femoropatellar joint arthrocentesis into K2-EDTA-treated bottles (K2-EDTA group), lithium-heparin-treated bottles (heparin group), and plain bottles (control group). The pH was determined using an electronic bench pH meter. The total nucleated cell count (TNCC) of samples was determined by hemocytometer method, while total protein (TP) concentrations, and lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities of the samples were determined spectrophotometrically at 2, 8, 24, 48, and 168 hours postcollection while being maintained at approximately 4°C. TP concentrations in the anticoagulant-treated groups remained stable for 48 hours. TNCCs were stable for 8 hours. However, after 2 hours, ALP, LDH, and pH varied significantly (P < 0.05). At 2 hours, mean ALP and LDH activities were significantly elevated in the lithium-heparin treatment samples, while the activity of these analytes was similar in the K2-EDTA and control groups. At 8 hours, the TNCC and pH were significantly elevated in K2-EDTA treated groups, while values were similar in lithium-heparin and control groups. No significant variation was seen in TP values at 2 hours, irrespective of treatment. The analytes-except for TP-became unstable within a few hours postcollection. Lithium-heparin and K2-EDTA treatments significantly altered ALP, LDH, TNCCs, and pH but not the TP concentrations of equine SF. Studies establishing reference intervals for these analytes based on the anticoagulant used are warranted to limit misinterpretations in clinical or research settings.

Detection of Leptomeningeal Carcinomatosis by Cerebrospinal Fluid in a Dog with a Negative MRI

ABSTRACT An 11 yr old female French bulldog was presented for acute onset of seizures and a 2 wk history of disorientation. On physical examination, a nodular mass at the fourth mammary gland level was observed. Neurological evaluation showed obtundation and compulsive behavior. Brain MRI study did not reveal any abnormalities. Cerebrospinal fluid (CSF) collected from the cerebellomedullary cistern showed a marked increase of total nucleated cell count (400 cells/μL). Cytological evaluation identified the presence of a monomorphic round cell population characterized by large cell bodies, a single eccentrical located nucleus with high nuclear:cytoplasmatic ratio, and marked atypia with anisocytosis, anisokaryosis, and multiple nucleoli. Leptomeningeal carcinomatosis (LC) was suspected. The dog was euthanatized for worsening of clinical signs. Post-mortem examination identified an anaplastic mammary carcinoma in the nodular mammary mass. Infiltration by neoplastic cells exhibiting the same morphological features was detected along leptomeninges of the telencephalon and cerebellum associated with cortical and subcortical parenchymal micrometastases. To our knowledge, this is the first case of LC in a dog detected by CSF evaluation but without any MRI abnormalities. This finding emphasizes the usefulness of CSF cytology in patients with suspected LC even in the absence of any MRI identifiable lesions.

Initial and follow-up evaluations on cerebrospinal fluid involvement by hematologic malignancy.

Central nervous system (CNS) involvement is a serious complication in hematologic malignancy, and early detection and management of CNS involvement in these cases significantly impact the prognosis. Currently, there is no consensus on the use of multiparametric flow cytometry (MFC) and conventional cytology (CC) testing for initial and follow-up cerebrospinal fluid (CSF) specimens to diagnose CNS involvement by hematologic malignancy. In our institution, after initial MFC and CC, two subsequent negative MFCs are required before discontinuing MFC. The aim of this study is to evaluate the outcome of this approach. CSF cytology and MFC reports were retrieved from Laboratory Information System, and data was reviewed. Between January 2020 and December 2021, 1789 CSF samples from 280 patients were submitted for CSF analysis. For those 517 CSF samples tested by both MFC and CC, 97 cases tested positive by both MFC and CC with 95% concordance. Eighteen cases were MFC + /CC - and 7 were MFC - /CC + . Thirty-six cases had initially positive MFCs followed by more than one MFC evaluation. Among those 36 cases, 22 cases (61.1%) converted to negative after the second follow-up sample, 9 cases (25%) were continuously positive for at least three samples, and 5 cases (13.9%) exhibited negative to positive conversion. Compared to negative CSF cases, positive CSFs had higher total nucleated cell count and higher total protein levels while red blood cells, glucose, and lactate dehydrogenase levels remained at comparable levels. The concordance between MFC and CC was excellent. The high incidence of positive MFCs on two or more follow-up samples and the high frequency of negative MFC to positive conversion indicate the necessity of repeated negative MFCs before discontinuing MFC. The fact that more than half of the positive cases converted to negative after the second CSF specimen and most follow-up positive cases can be detected by CC alone suggests it is adequate to use CC alone for follow-up CSF study after two consecutive negative MFCs.

Intrabone Transplantation of a Single Unwashed Umbilical Cord Blood Unit with Antithymocyte Globulin-Free and Sirolimus-Based Graft-versus-Host Disease Prophylaxis: Fast Immune Reconstitution and Long-Term Disease Control in Patients with High-Risk Diseases

Several strategies have been explored with the attempt of improving the safety and feasibility of umbilical cord blood transplantation (UCBT) in adults. The aim of this retrospective analysis was to examine the safety and efficacy of intrabone transplantation of a single unwashed cord blood unit in an antithymocyte globulin-free, sirolimus-based graft-versus-host disease prophylaxis platform. We collected data for all consecutive UCBTs infused intrabone (IB) and unwashed at San Raffaele Hospital in Milan between 2012 and 2021. Thirty-one consecutive UCBTs were identified. All but 3 UCB units had a high-resolution HLA typing on 8 loci at the time of selection. At the time of cryopreservation, the median CD34+ cell count was 1 × 105/kg (range, .6 to 12.0 × 105/kg) and the median total nucleated cell (TNC) count was 2.8 × 107/kg (range, 1.48 to 5.6 × 107/kg). Eighty-seven percent of patients received myeloablative conditioning, and 77% underwent transplantation for acute myeloid leukemia. The median duration of follow-up among survivors was 38.2 months (range, 10.4 to 123.6 months). No adverse events were related to the IB infusion at bedside under short-conscious periprocedural sedation or to the no wash technique. After thawing, median CD34+ cell and TNC counts were .8 × 105/kg (range, .1 to 2.3 × 105/kg) and 1.42 × 107/kg (range, .69 to 3.2 × 107/kg). The median time to engraftment was 27 days for neutrophils and 53 days for platelets. One patient experienced graft rejection and was subsequently rescued with a salvage transplantation. The median time to a CD3+ cell count >100/μL was 30 days. The 100-day cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 12.9% (95% confidence interval [CI], 4% to 27.3%), and the 2-year cumulative incidence of moderate-to-severe chronic GVHD (cGVHD) was 11.8% (95% CI, 2.7% to 28.3%). At 2 years, overall survival (OS) was 52.7% (95% CI, 33% to 69%), relapse incidence was 30.7% (95% CI, 13.7% to 49.6%), and transplantation-related mortality was 29% (95% CI, 14.3% to 45.6%). In univariate analysis, infused CD34+ cell count did not impact transplantation outcomes. In patients who underwent transplantation in first complete remission, relapse rate was 13%, with a 2-year OS >90%. In our cohort, IB infusion of a single cord blood unit was feasible, with no adverse reactions related to the no wash/IB infusion, low rates of cGVHD and disease relapse, and rapid immune reconstitution.