Buffy Coat Preparations Research Articles

Biochemical characterization and discrimination of Leishmania major parasites and infected macrophages with Raman spectroscopy and chemometrics

Leishmaniasis is classified as one of the neglected tropical disease (NTD), which are caused by a group of parasitic protozoans called Leishmania. The high case load and severity of the disease make Leishmaniasis second only to malaria in terms of both severity and infectivity. However, due to the low economic interest in research and development, it may become a major world-wide health threat. Current diagnostics including serological assessment of infected tissue by either light microscopy, or antibody tests or by the culturing of potential infection via in vitro or in vivo animal inoculation, parasitological tests using samples aspirated from the spleen and bone marrow, Immunological tests such as the Montenegro test, Fluorescence assays, and polymerase chain reaction (PCR) techniques are suffer from several limitations including time and expense. Herein, we first apply Raman microscopy to distinguish the two L. Major parasitic forms namely promastigotes and amastigotes and secondly, distinguish infected from non-infected macrophages using multivariate data analysis including Principal Component Analysis (PCA) and unsupervised hierarchical cluster imaging analysis (UHCA). The maximum variance between infected and uninfected macrophage groups are visible in the lipid region (92.20 %) as compared to the fingerprint region (46.13 %) along PC1. The contributions from nucleic acids can be found at 805 cm−1 (phosphodiester - Z-marker), 767 cm−1 (pyrimidine ring breathing mode), 742 cm−1 (ring breathing mode of DNA/RNA bases), and 568 cm−1 (cytosine/guanine). These amplified nucleic acid signals in infected macrophages indicate the presence of infection compared to the uninfected macrophage group. Similarly, the maximum variance between amastigotes and promastigotes groups are observed in the lipid region (88.45%) as compared to the fingerprint region (28.34 %). Moreover, the UHCA of infected macrophages revealed the accumulation of lipid bodies or droplets inside or close proximity parasitophorous vacuole, which is consistent with the reported literature. Once established macrophages were infected with Leishmania in vitro and the differences between infected and non-infected established with high reproducibility. The reported spectral differences between infected and non-infected macrophages lays the ground work for developing a diagnostic tool for detection of leishmaniasis in a buffy coat preparation and also offers the potential of monitoring the effects of new therapeutics.

Clinical Validation of a Circulating Tumor Cell Assay Using Density Centrifugation and Automated Immunofluorescence Microscopy.

The US Food and Drug Administration (FDA)-approved CELLSEARCH assay (Menarini Silicon Biosystems) for circulating tumor cells (CTCs) relies on expression of an epithelial cell adhesion molecule to enrich for CTCs. We sought to validate a CTC assay (RareCyte) for clinical use that instead collects a buffy coat preparation enriched for CTCs. Normal peripheral blood specimens spiked with cultured breast and prostate cancer cells and 47 clinical samples were used to validate assay performance. Specimens were enriched for buffy coat cells and applied onto 8 glass slides. The slides were immunofluorescently stained and imaged by automated microscopy and computer-aided image analysis. The assay was 100% specific for detecting spiked tumor cells. For samples spiked with 25, 50, and 125 cells, the percentage coefficients of variation were 42%, 21%, and 3.7%, respectively. Linearity studies demonstrated a slope of 0.99, an intercept of 1.6, and R2 of 0.96. Recoveries at the 25-, 50-, and 125-cell levels were 92%, 111%, and 100%, respectively. Clinical samples run on both CELLSEARCH and RareCyte correlated with an R2 of 0.8 after log-transformation and demonstrated 87.5% concordance using the CELLSEARCH criteria for predicting adverse outcomes. The RareCyte CTC assay has comparable performance to the FDA-cleared method and is ready for further clinical validation studies.

Short- and long-term reproducibility of the COMET assay for measuring DNA damage biomarkers in frozen blood samples of the EPIC-Heidelberg cohort

The comet assay is widely used for quantification of genomic damage in humans. Peripheral blood derived mononuclear cells (PBMCs) are the most often used cell type for this purpose. Since the comet assay can be performed in an enhanced throughput format, it can be applied to large sample collections such as biobanks. The European Prospective Investigation into Cancer and Nutrition (EPIC) study is one of the largest existing prospective cohort studies, and the German Cancer Research Institute (DKFZ) in Heidelberg is a participating center with 25.000 frozen blood samples stored from around 25 years ago, enabling retrospective assessment of disease risk factors. However, experience with decades long frozen samples in the comet assay is so far missing. In Heidelberg, 800 study participants were re-invited twice between 2010 and 2012 to donate further blood samples. Here, we analyzed 299 Heidelberg-EPIC samples, compiled from frozen PBMC and buffy coat preparations selected from the different sampling time points. In addition, 47 frozen PBMC samples from morbidly obese individuals were included. For buffy coat samples, we observed a poor correlation between DNA damage in the same donors assessed at two sampling time points. Additionally, no correlation between DNA damage in buffy coat samples and PBMCs was found. For PBMCs, a good correlation was observed between samples of the same donors at the two time points. DNA damage was not affected by age and smoking status, but high BMI (>30; obesity) was associated with increased DNA damage in PBMCs. There was no indication for a threshold of a certain BMI for increased DNA damage. In conclusion, while 25 year-long stored buffy coat preparations may require adaptation of certain experimental parameters such as cell density and electrophoresis conditions, frozen PBMC biobank samples can be analyzed in the comet assay even after a decade of storage.

Ketotifen is a Prodrug. Norketotifen is the active metabolite.

Evaluation of the in vitro human liver microsome and hepatocyte metabolism of ketotifen demonstrated that norketotifen (NK) is the major demethylated hepatic metabolite of ketotifen. It is here reported that NK is completely devoid of the severe and dose-limiting sedative effects of ketotifen. Thus, while ketotifen is clinically dose-limited to 1mg, bid, there are no dose-limiting sedative effects elicited by NK, even after the highest single-dose (16 mg) or after repeat-doses (8mg × 7 days) in humans or after the highest doses given to dogs in repeat-dose toxicological studies (40 mg/kg × 14 days). In addition, NK-but not ketotifen-was found to express potent and dose-dependent inhibition of the release of the pro-inflammatory cytokine TNFα from activated human buffy coat preparations. Thus, when used as an anti-inflammatory drug, ketotifen is the sedating prodrug which is converted to NK a nonsedating metabolite with anti-inflammatory activity.

Isolation and identification of peste des petits ruminants virus from goats in Egyptian governorates.

Background and Aim:The peste des petits ruminants (PPR) is a highly contagious disease of small ruminants which negatively affects animal production and the socioeconomic status of farmers. Peste des petits ruminants virus (PPRV) encodes eight proteins, with the viral fusion protein (F) playing a role in virus virulence and stimulating an effective protective immune response. This study aimed to isolate and complete the identification of PPRV circulating in goats in different Egyptian governorates and perform molecular characterization of the PPRV F gene.Materials and Methods:Samples were collected from unvaccinated animals with clinical signs suggestive of PPR. A total of 256 sera were tested for the detection of PPRV antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA) kit, while 214 samples of blood buffy coat preparation, animal swabs (nasal, ocular, and saliva), and fecal and tissue samples were tested for the detection of the PPRV antigen using an antigen-capture ELISA kit. Molecular diagnosis, gene cloning, blast analysis, and phylogenetic analysis were performed for the molecular characterization of PPRV.Results:The seroprevalence results of PPRV antibodies in the tested sera showed a total of 67.9% positive samples. The rates of PPR antigen recorded by the antigen-capture ELISA in the swabs (nasal and ocular) and tissue samples were 44.3%, 46.8%, and 43.5%, respectively, with saliva swabs having the highest rate of PPRV positivity (76.4%) and fecal samples having the lowest (33.3%). Molecular characterization of the PPRV Vero cell culture revealed that the circulating PPRV strain belongs to the IV lineage. Blast analysis of the PPRV F gene showed 96.7% identity with the PPRV strain Egypt-2014 fusion protein (F) gene, KT006589.1, differing by 43 single-nucleotide polymorphisms.Conclusion:The results of this study indicate that the emerging PPRV belongs to the IV lineage among small ruminant animals. The findings also indicate the need for an innovative strategy to control and eliminate this disease based on a regularly administered and effective vaccine, a test to distinguish between infected and vaccinated animals, and the need for further study on the protein structure and PPRV F gene expression, which should help us to understand the molecular evolution of the virus and control and eliminate PPR disease.

Abstract B31: Detection of viable tumor cells from cryopreserved buffy coat using the VTX-1 Liquid Biopsy Platform

Abstract Introduction: Several circulating tumor cell (CTC) technologies have been developed amidst growing interests in the “liquid biopsy” field. The fully automated VTX-1 platform is developed to gently isolate intact CTCs from whole blood with high recovery and viability, compatible with many applications (1-3). However, many CTC technologies require processing of fresh patient samples at off-site facilities. Here we present encouraging preliminary results showing viable cell isolation by VTX-1 from cryopreserved buffy coat, using a telomerase-selective replicating adenovirus (TelomeScan). This widens potential applications of viable CTC detection for retrospective clinical studies and drug development. Methods: Buffy coat preparation: Buffy coat was prepared by centrifugation of healthy donor blood collected in EDTA, heparin, or heparin CPT vacutainers. Cell spiking and processing: 1) Cancer cells were spiked into freshly prepared buffy coat and processed with the Vortex chip at different dilutions. 2) Cancer cells were labeled and spiked into healthy donor blood, and buffy coat was prepared and processed by VTX-1 either immediately or following cryopreservation up to 1 week. Imaging was evaluated for cell recovery. Viable, telomerase-activated tumor cell detection: VTX-1 enriched cells were directly collected in chamber slides and infected with the TelomeScan adenovirus (OBP-401) for 24 h (4). The percentage of GFP (+), viable, telomerase-expressing cells was evaluated alongside cell recovery. Results: MCF7 cells were spiked into freshly prepared buffy coat, resuspended in 8mL, and processed at 5X, 10X, and 20X dilutions. The best compromise between recovery, WBC count, and processing time was obtained for the 10X dilution with 46% recovery. The majority of spiked cells (MCF7 and H1299) collected from buffy coat were shown to be viable, as demonstrated by infection with the telomerase-selective replicating adenovirus (TelomeScan, OBP-401). Cryopreservation of the buffy coat samples showed a slight reduction following cryopreservation. Discussion: The optimal performance of the Vortex chip on buffy coat samples has previously been shown at 10X dilution. This capability of the VTX-1 was further demonstrated through infection with a telomerase-selective replicating adenovirus. This combined workflow has shown seamless integration from whole blood. Noteworthy recovery and sensitivity were also observed with cryopreserved buffy coat. Patient biobanked samples are currently being pursued. The overall advantages are: a) viable cell detection, b) direct collection of cells into culture media ready for in vitro testing, c) no requirements for fixation, labeling, backflushing, d) a very low WBC background eliminating false positives, and e) the potential to retrospectively analyze clinical biobanked samples at a centralized laboratory.

Prevention of Panton-Valentine Toxin-Mediated Leucocyte Destruction Depending on the Ultra-Diluted Staphylococcinum Potency Gradient

Abstract Background Panton-Valentine toxin (PVT) is an important pathological marker of staphylococcal infection mediated by functional as well as morphological damage of the phagocytic cells. Human body being an ecological niche for the bacteria shows higher affinity toward staphylococcal infection. A steady escalation in mortality and morbidity associated with antibiotic resistance in gram-positive infections is an emerging threat all over the globe; thus, it is important to find out an alternative strategy that can diminish the virulence and pathogenicity of the bacteria. Staphylococcin is a colicin-type chemical secreted by Staphylococcus aureus helps to prevent growth of organisms other than its progenitor. In this study, we evaluated the efficacy of homoeopathic medicine Staphylococcinum against staphylococcal PVT at different potencies (6CH, 12CH, 30CH and 200CH). Materials and Methods Different potencies of Staphylococcinum were administered in a leucocyte buffy coat preparation infected with staphylococcal suspension (0.5 McFarland's standard) along with control with alcohol. They were kept in incubator for 2 hours and then centrifuged at 1200 rpm for 5 minutes. Smears prepared on slides with centrifuged deposits stained by Preston and Morrell's modified Gram's method of staining and evaluated under the microscope. Results It was observed that there was extensive destruction of leukocytes in control and 6CH potency, while the degree of destruction decreased markedly from 12CH to 200CH. At 200CH potency, leukocytes were almost normal, which clearly indicate the preventive action of Staphylococcinum against PVT. The mean percentages of intact leucocytes were 0.73, 0.93, 10.00, 27.67 and 65.00 in control, 6CH, 12CH, 30CH and 200CH potencies, respectively. Conclusion The finding may help in the use of this medicine in moribund patients in cases of disseminated S. aureus infection as there is no known side effect of the medicine. However, in vivo study is necessary before such use in those cases.

Le mélange de concentré de granulocytes de sang total (MCGST) comme alternative au concentré de granulocytes d’aphérèse (CGA)

BackgroundThe collection of granulocytes by apheresis requires volunteer donor stimulation by corticoids and the use of HES, a compound which is currently challenged by potential safety issues. Preparation of pooled granulocytes concentrates from whole blood buffy coats (PGC) represent an alternative to apheresis with a better benefit/risk for the donors. MethodWhole blood is collected in a bottom and top blood bag for buffy coat preparation. After centrifugation and separation, buffy coat are obtained. Twenty ABO matched buffy coats are selected for processing into one PGC. Four pools of five buffy coats were made, platelet additive solution is added to each pool, mixed gently and centrifuged. The red cell residue, supernatant and granulocyte rich layer are separated. Two granulocyte rich layers are pooled and added with 70mL of ABO matched plasma from the initial donations (=PGC10). The final PGC (=PGC20) is obtained by pooling two PGC10 into a platelet storage bag. Neutrophil content and in-vitro functionality are assessed at day of preparation (D1) and at expiry hour, 48 hours after collection (D2). ResultsOn N=18, mean: Volume=408±4mL, 2.2*1010±0.24 neutrophils, Hematocrit=18%±3%, 4.7*1011platelets. Viability is well preserved: 95%±6% day of PGC preparation, 85%±7% after 24h of storage (D2). Functionality (ROS production measurement) is well preserved: 1.36±0.25 at D1 and 1.38±0.18 at D2. Expression and modulation of adhesion molecules after stimulation are normal at D1 and slightly decreased at D2 but still normal. ConclusionsPGC20 in vitro characteristics are in conformance with the EDQM guide (V19) and similar to apheresis for granulocytes content and hematocrit. The viability and two mean indicators which explore neutrophil function are well maintained during PGC preparation and after 24 hours of storage.

An automated mini buffy coat preparation method for use in mini extracorporeal photopheresis treatment of graft-vs-host-disease in a low body weight pediatric patient.

A mini extracorporeal photopheresis (mini-ECP) "off line" technique has been developed for use in the treatment of small children and patients with apheresis contraindications. Until now various methods have been used for buffy coat separation from whole blood. In this report we describe a protocol for mini buffy coat preparation using the automated Sepax laboratory separator for "off line" ECP treatment in a low body weight child with graft-vs-host-disease. According to our results this alternative method has been proven feasible and tolerable.

Dilemma in Diagnosis of Pyogenic Meningitis in Cerebrospinal Fluid Contaminated with Blood: Does Leucocyte Esterase Test Help?

Context:Pyogenic meningitis is often a devastating condition which is diagnosed by analysis of cerebrospinal fluid (CSF) obtained by lumbar puncture (LP). CSF thus obtained can get contaminated with peripheral blood leucocytes during LP which renders it unusable for cytological analysis. Leucocyte esterase strips are available for identifying leucocyte esterase activity in urine and other body fluids which suggest inflammation. We conducted this experiment to see whether the leucocyte esterase strip can differentiate between neutrophils invited at the inflammatory site and circulating neutrophils in CSF.Aim:To compare the diagnostic ability of the leucocyte esterase test between pyogenic meningitis and CSF contaminated with circulating neutrophils.Setting and Design:Prospective analytical study conducted in a tertiary care hospital.Materials and Methods:The CSF samples of pyogenic meningitis patients were analyzed for leucocyte esterase activity. The other group was normal CSF which was deliberately contaminated with buffy coat preparation, and leukocyte esterase activity was determined.Statistical Analysis:Diagnostic ability of a test in terms of sensitivity and specificity.Results:Overall sensitivity of the dipsticks in diagnosing pyogenic meningitis is 81% and specificity is 99%. When compared with experimentally contaminated CSF sample, a reading of 2+ on the strip had a sensitivity of 70% and specificity of 100% for pyogenic meningitis.Conclusion:Leucocyte esterase strip is specific for pyogenic meningitis (activated neutrophils), and hence can differentiate from CSF contaminated with blood.

A Survey of Trypanosome Species Circulating In Pigs In Three Selected Local Government District of Enugu State, Nigeria

A cross sectional survey of trypanosomosis of pigs was carried out in three randomly selected Local Government Areas (Nsukka, Udenu and Igbo Eze South) out of the six local government areas in Enugu North Senatorial Zone. Blood samples were randomly collected from a total of 1800 pigs from abattoirs and farms and were examined for trypanosomes parasites using standard parasitological methods: wet film, buffy coat and Giemsa-stained thin smear preparations. A total of 93 (5.2%) pigs were positive overall. Among the positive samples, Trypanosoma brucei was the predominant species (86.0%), followed by T. congolense (13.9%). The different relative isolation rate for Nsukka, Igbo-Eze South and Udenu Local government areas were 14 (4.7%), 14 (4.7%), and 19 (4.0%) respectively in the dry season, and 17 (5.7%), 17 (5.7%) and 12 (6.3%) in the rainy season respectively. The trypanosome prevalence in males from Nsukka, Igbo-Eze South and Udenu local governments were 6.5%, 6.2% and 4.2% respectively, while report in female were 3.8%, 4.1% and 5.9% in the three local governments respectively. There was no significant variations (p<0.05) with seasons, sex and age groups. It was therefore concluded that porcine trypanosomosis is still endemic in Enugu North Senatorial Zone with T. brucei being the most incriminated as the cause of the disease in the zone.

Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n=10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions.

Correlation between Epstein-Barr Virus Infection and Disease Activity of Systemic Lupus Erythematosus: a Cross-Sectional Study

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease for whose pathogenesis viral infections are important. The Epstein-Barr virus (EBV) is the main infectious etiological agent. This study aimed to quantitative evaluation of EBV in SLE patients.Materials and Methods:In this cross-sectional study, 40 patients with SLE diagnosed based on American College of Rheumatology criteria were selected using purposive sampling. All were included in the study after obtaining informed consent for participation. Whole blood samples were taken and buffy coat preparations were isolated to determine viral load using the real-time polymerase chain reaction method and assessment with the SLE disease activity index (SLE-DAI).Results:From a total of 40 patients, 37 cases (92.5%) were women. The EBV test was positive in 67.5% and mean viral load was 5396 ± 1891.9 copy/ml. Twenty of forty patients had active and 50% inactive disease, mean EBV viral loads being 6798 and 28.25 copy/ml, respectively (P-value = 0.003). In terms of the severity of disease activity, 17.5 % of female patients had mild to moderate activity, whilst 32.5% of them had severe activity, with respective viral loads of 5,803.3 and 29.73 copy/ml (P-value = 0.003).Conclusion:The Epstein-Barr viral load in SLE patients with active disease was found to be markedly higher than in inactive cases. Thus, EBV may have an important role in the pathogenesis and activity of SLE.

Erythrocyte Sialoglycoproteins Engage Siglec-9 to Maintain Neutrophil Quiescence in the Bloodstream

Neutrophils remain functionally quiescent in the bloodstream of healthy humans. Characterized by a short lifespan (for which estimates vary widely), they exit the bloodstream to mediate various functions such as pathogen elimination or wound healing--or undergo spontaneous apoptosis and macrophage-mediated clearance in the absence of an inflammatory stimulus. Understanding of neutrophil quiescence, activation and lifespan is relevant to many clinical situations such as infection, sepsis, reperfusion injury, granulocyte transfusions, and other neutrophil-mediated inflammatory pathologies.It has long been known that neutrophils purified from blood tend to be short-lived and show signs of activation, but this technical challenge has been assumed to be a reflection of their natural biology in vivo. We found that when purified from whole blood, neutrophils indeed rapidly express activation markers (low L-selectin; high CD11b) and progress to apoptosis. However, using a novel method to label neutrophils in situ followed by flow cytometry, we found that these activation events occurred at a much slower rate in neutrophils that remained in whole blood. This finding led us to hypothesize that during separation of neutrophils from other blood components, one may be removing an inhibitor that normally maintains neutrophil quiescence in the bloodstream.Co-incubation studies showed that erythrocytes are the primary blood component that dampens neutrophil activation, including chemotaxis, generation of reactive oxygen species, and release of neutrophil extracellular traps. The duration of neutrophil viability was also lengthened upon re-incubation with erythrocytes. We found that this maintenance of functional quiescence is mediated largely by erythrocyte surface sialoglycoproteins (particularly glycophorin A, GPA), which engage neutrophil Siglec-9, a sialic acid-binding receptor that is known to dampen innate immune cell activation via cytosolic inhibitory motifs. Modification of erythrocyte sialic acid side chains using a newly developed gentle method eliminated Siglec-9 binding, and allowed neutrophil activation as measured by reactive oxygen species production.We next sought evidence that this interaction occurs in vivoby studying freshly collected whole blood. Smears made from fresh whole blood showed a high degree of Siglec-9 clustering on neutrophils, which was evident at points of contact with GPA on erythrocytes. This clustering was markedly reduced when smears were made from buffy coat preparations, which involves initial physical separation of neutrophils from erythrocytes during centrifugation.Taken together, this data indicates that erythrocyte sialic acids have an unexpected function as carriers of "self-associated molecular patterns" (SAMPs), regulating innate immunity and maintaining neutrophil quiescence in the bloodstream, apparently by tonic engagement of inhibitory Siglec-9. Notably, this SAMP effect blunts but does not completely inhibit bacterial killing by neutrophils in whole blood, and yet presumably helps to limit unwanted neutrophil inflammatory activation in the bloodstream. Our findings are relevant to many physiological, pathological and clinical situations involving neutrophil biology, and may be useful in reevaluation of prior studies of activation, function and reinfusion-based kinetics that were performed using neutrophils isolated away from whole blood. DisclosuresNo relevant conflicts of interest to declare.

IMST-12. INVESTIGATION INTO MECHANISMS OF IMMUNOSUPPRESSION IN GLIOMA

Glioblastoma induces immunosuppressive effects by several, redundant mechanisms, TGF-β/VEGF signalling pathway, IL-2Rα/CD25-mediated, CTLA-4/PD-1/PD-L1 immune checkpoint interaction and STAT3 activation. All of these mechanisms have been investigated as potential therapeutic targets for glioblastoma treatment in various clinical and preclinical trials. Tregs are a subset of CD4+ T cells that infiltrate tumours. They suppress proliferation and activity of effector T cells. Treg frequency increases with increasing WHO grade. Tregs and effector T cells express co-inhibitory PD-1 receptor and in melanoma can be divided into 3 compartments by expression levels of FoxP CD45RA and CD25;1. FoxP3(lo)CD45RA+CD25(lo) – naïve Treg cells,2. FoxP3 (hi)CD45RA-CD25(hi) – highly suppressive,3. FoxP3 (lo)CD45RA-CD25(lo) – non-suppressive. Serum from 34 high grade and 34 low grade glioma patients, were analysed for the concentration of TGF-β, PD1 and its ligand PD-L1 along with 33 control sera from non-cancerous patients using an ELISA technique. Tissue samples embedded in paraffin wax from the same patients were also examined for the presence of IL-2Rα using immuno-histochemistry. Treg cells from these patients were isolated from buffy coat preparations and were examined using a flow cytometric technique. There were significant differences in the measured analytes between sera of glioma patients of low and high grade and non-cancerous control patients. Tumour tissue also revealed increased expression of the IL-2 receptor. Treg cells displayed compartmentalisation into several subtypes. These results suggest that specific protein levels can be used to detect and confirm a glioma diagnosis as well as to distinguish between low and high grade gliomas. These results may also be used to predict tumour recurrence and transformation between low and high grade gliomas. There was an interesting up-regulation of the IL-2 receptor on tumour cells which indicates that these cells may have a role in immunosuppression and immunosurveillance.

An alternative mini buffy coat preparation method for adult patients with extracorporeal photopheresis contraindications.

Extracorporeal photopheresis (ECP) is an important cell-based therapy for various diseases but is limited to patients eligible for apheresis. We developed an alternative mini buffy coat (BC) preparation method using the Spectra Optia® apheresis system and compared its efficacy of white blood cell (WBC) recovery with the standard mini BC preparation method already established for pediatric patients. Whole blood (450 ± 45 mL) samples were collected from 30 randomly selected healthy volunteer blood donors and divided into two groups. In the first group, WBCs were separated with a fully automated separator device (Compomat G4® ). In the second group, BCs were separated with the bone marrow processing program of the Spectra Optia apheresis system. There were no significant differences in total leukocyte counts per product between the two groups. In contrast, lymphocyte counts per product were significantly higher (P < 0.001) in BCs separated from apheresis. Our novel technique resulted in similar WBC yields but higher lymphocyte yields than the standard mini BC preparation method. This method can serve as an alternative to WBC collection in conventional ECP for adult patients with apheresis contraindications. J. Clin. Apheresis 32:12-15, 2017. © 2016 Wiley Periodicals, Inc.

Ultrastructure of Theileria-infected blood cells of loggerhead turtles

The authors are grateful to Drs. Stacy and Harvey et al. for their comments concerning Caretta caretta theileriosis. It gives us an opportunity to clarify and expand our statements as well as to offer further micrographic evidence relative to understanding this condition. This report regarding theileriosis is based on observations that were made over a period of about 12 years and includes an analysis of multiple hematological parameters derived from a large number of C. caretta obtained from inhabitants of the Mediterranean Sea (see Basile et al. 2012). In spring 2011, two specimens of C. caretta, completely covered by leeches, were brought to the Turtle Center of the Zoological Station of Naples. The leeches were recognized as Ozobranchus margoi, well known as turtle leeches (see figure in Di Santi et al. 2012). The diagnostic process of theileriosis began with the detection of eosinophilic leukocytosis (Fig. 1a, Di Santi et al. 2012, and here in Fig. 1). It is well acknowledged that eosinophilia is a very frequent manifestation of parasitic infection in vertebrates. The first notable observation that was made was the presence of bacillary rodbayonet-shaped inclusions in erythrocytes (Bthe most useful guide to the genus Theileria,^ Kreir 1977) followed by their recognition in leukocytes. An abundant number of techniques were utilized to identify the parasite. The first step was to conduct weekly hemograms on the two specimens which embraced morphologic analysis of blood films for parasitized cells Figs. 2–7, 8–9, 10–12, and 13). In addition, samples were preserved in EPON preparations to study them under TEM (see enclosed unpublished photographs of Figs. 14–15, 16–17, 18–19, 20–21, 22–25, and 26–27). During the stage of infection, osmotic fragility tests were conducted to establish whether the parasitic infection resulted in an alteration of erythrocytic osmotic fragility. When the stage of infection visually affected the leukocytes in the blood smears, buffy coat preparations of both subjects were prepared and cultured. The results of these tests confirmed the presence of an

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Evaluation of Leukocyte and Bacterial Interference in Point-of-Care Human Chorionic Gonadotropin Tests

We read with interest the Letter to the Editor by Jao et al. [1] which reported false positive results using the point-of-care (POC) One Step Pregnancy Test device (ACON Laboratories Inc., San Diego, CA, USA). The authors determined that false positive results occurred when the concentration of white blood cells (WBCs) in a urine specimen exceeded 0.4×109 cells/L. We recently observed several false-positive human chorionic gonadotropin (hCG) results using the POC hCG Combo Rapid Test (Cardinal Health, Waukegan, IL, USA). We observed that most of our false positive specimens were in patients with evidence of urinary tract infection (UTI) and pyuria; with this information, and in the context of the findings by Jao et al. [1] that WBCs may result in false positive hCG assays, we evaluated the effect of WBCs or bacteria in urine on the analytical performance characteristics of both devices. Two buffy coat preparations were created using pooled whole blood from 25 patients. The buffy coat preparations were analyzed using the Coulter LH 750 Hematology Analyzer (Beckman Coulter, Brea, CA, USA) to assess the purity of the preparation. WBCs were prepared on two separate occasions and the total WBC counts of the pools were 57.5×109/L and 200×109/L. These WBC preparations were then diluted into urine from a male patient at six different WBC concentrations (0.1×109, 0.4×109, 1.0×109, 5.0×109, 10.0×109, and 21.0×109/L) including and exceeding the concentration used by Jao et al. [1]. The urine was then tested on both the hCG One Step and the hCG Combo Rapid test according to the manufactures' recommendations. All specimens were negative on both devices. The explanation for the different results observed in the study by Jao et al. [1] and the current study are unclear. Jao et al. [1] do not describe the source of their WBC preparation, it is unknown if it is from one patient or multiple patients, and the WBC differential is also unknown. It is possible that differences in these parameters could explain the discrepancy. With the knowledge that most of our patients with false positive results had evidence of UTI, and knowing that Escherichia coli is the most common cause of UTI in the outpatient setting, we wondered if E. coli specifically might account for the false positive results. To test this hypothesis, we made logarithmic dilutions (107 colony forming units [CFU]/mL, 106 CFU/mL and 105 CFU/mL) of a clinical isolate of uropathogenic E. coli into sterile urine from a male patient and tested these samples on both devices; all tests yielded negative results. To account for the fact that addition of E. coli and/or WBCs to sterile urine might not accurately replicate the matrix of a clinical specimen from a patient with UTI, we then tested 10 urine specimens from unique, female patients that were culture positive for E. coli (>50,000 colonies/mL) and 5 additional urine specimens that were submitted from outpatients to the microbiology laboratory for culture for presumed UTI. All of these specimens, when tested with the hCG Combo Rapid Test, also gave negative results. A recent study by Snyder et al. [2] reported that high WBC concentrations resulted in falsely increased serum hCG concentrations using the Beckman Coulter Access Total βhCG on the UniCel DxI (Beckman Coulter, Chaska, MN, USA). According to the authors, the false positive results may be attributed to leukocyte alkaline phosphatase reacting with the conjugate that is used in those immunoassays. However, both POC devices used in this particular study use colloidal gold as the color conjugate, hence leukocyte alkaline phosphatase should not result in this specific interference. Based on the information in this study, we feel there is inadequate evidence at this time to conclude that false-positive hCG results, in either the One Step Pregnancy Test device or the hCG Combo Rapid Test, can be attributed to elevated WBC concentrations in the urine. However, we continue to see false positive results that suggest the presence of a yet unidentified interfering substance.

The Signal Peptide of the Tumor-shared Antigen Midkine Hosts CD4+ T Cell Epitopes

The CD4 T cell response to the tumor antigen Midkine was unknown. Most of the T cell response to Midkine relies on T cell epitopes contained in its signal peptide. The signal peptide of Midkine is accessible to HLA class II pathway for CD4 T cell presentation. It is a new function for signal peptides to contribute to tumor-specific CD4 T cell response. Because of the key role of CD4 T cell response in immunity to tumors, we investigated the CD4(+) T cell response to the recently identified tumor antigen Midkine (MDK). By weekly stimulations of T lymphocytes harvested from seven HLA-DR-typed healthy donors, we derived CD4(+) T cell lines specific for eight MDK peptides. Most of the T cell lines reacted with the peptides 9-23 and 14-28, located in and overlapping the MDK signal peptide, respectively. Accordingly, the MDK signal peptide appeared to be rich in good binders to common HLA-DR molecules. The peptide 9-23-specific T cell lines were specifically stimulated by autologous dendritic cells loaded with lysates of MDK-transfected cells or with lysates of tumor cells naturally expressing the MDK protein. One T cell line was stimulated by HLA-compatible MDK-transfected tumor cells. By contrast, the peptide 14-28-specific T cell lines were not stimulated in any of these conditions. Our data demonstrate that CD4(+) T cell epitopes present in the signal peptide can be accessible to recognition by CD4(+) T cells and may therefore contribute to tumor immunity, whereas a peptide overlapping the junction between the signal peptide and the mature protein is not.