Diagnosis and Monitoring of Cystinosis Using Immunomagnetically Purified Granulocytes

Abstract BACKGROUND Cystine determination is a critical biochemical test for the diagnosis and therapeutic monitoring of the lysosomal storage disease cystinosis. The classical mixed-leukocyte cystine assay requires prompt specialized recovery/isolation following blood drawing, providing cystine concentrations normalized to total protein from assorted types of white blood cells, each with varying cystine content. METHODS We present a new workflow for cystine determination using immunomagnetic granulocyte purification, and new reference ranges established from 47 patient and 27 obligate heterozygote samples assayed. Samples were collected in acid-citrate dextrose tubes and their stability was proven to allow for overnight shipping before analysis. Cystine was quantified by LC-MS/MS. RESULTS The new method was reproducible (<15% root mean square error) and specific, assaying purified granulocytes from blood samples that no longer required immediate preparation and therefore allowing for up to 30 h before processing. There was a nearly a 2-fold increase in the therapeutic target (1.9 nmol half-cystine/mg protein) range, established using distributions of patient, obligate heterozygote, and control samples. The 2.5–97.5 percentile ranges (−2 SD to +2 SD around mean) for these cohorts were 0.67–6.05 nmol/mg protein for patients, 0.33–1.35 nmol/mg protein for obligate heterozygotes, and 0.09–0.35 nmol/mg protein for controls. CONCLUSIONS The intracellular cystine determination method using immunopurified granulocytes followed by LC-MS/MS analysis improves the inherent variability of mixed leukocyte analysis and eliminates the need for immediate sample preparation following blood draw.

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Simple Tube Centrifugation Method for Platelet-Rich Plasma (PRP) Preparation in Catalonian Donkeys as a Treatment of Endometritis-Endometrosis

Animals ◽

10.3390/ani11102918 ◽

2021 ◽

Vol 11 (10) ◽

pp. 2918

Author(s):

Priscila Fantini ◽

Román Jiménez ◽

Karina Vilés ◽

Antoni Iborra ◽

Maristela Silveira Palhares ◽

...

Keyword(s):

Transforming Growth Factor ◽

Therapeutic Potential ◽

Platelet Rich Plasma ◽

White Blood Cells ◽

Fold Increase ◽

Enzyme Linked Immunosorbent Assay ◽

Platelet Concentration ◽

Wbc Count ◽

Citrate Dextrose ◽

Tgf Β1

The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets (2 or 3 times baseline physiologic values) and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-β1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-β1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.

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Conditions Affecting the Responses of Human Platelets to Epinephrine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647031 ◽

1988 ◽

Vol 60 (02) ◽

pp. 209-216 ◽

Cited By ~ 23

Author(s):

Chantal Lalau Keraly ◽

Raelene L Kinlough-Rathbone ◽

Marian A Packham ◽

Hidenori Suzuki ◽

J Fraser Mustard

Keyword(s):

Platelet Rich Plasma ◽

Shape Change ◽

Human Platelets ◽

Dense Granule ◽

Primary Phase ◽

Lag Phase ◽

Acid Citrate Dextrose ◽

Two Phases ◽

Citrate Dextrose ◽

Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

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RISTOMYCIN (AGGRISTIN) PRECIPITATION TEST: A NEW, RAPID AND RELIABLE METHOD FOR THE DETECTION OF INTRAVASCULAR CLOTTING

10.1055/s-0038-1643133 ◽

1987 ◽

Author(s):

G Pfliegler ◽

J Arnout ◽

J Vermylen

Keyword(s):

Reliable Method ◽

Degradation Products ◽

Laboratory Diagnosis ◽

Blood Collection ◽

Vein Thrombosis ◽

Fibrin Degradation Products ◽

Acid Citrate Dextrose ◽

Citrate Dextrose ◽

Precipitation Test

The rapid and specific detection of fibrin monomers (fm) and fibrin degradation products (fdp) is of major importance in the laboratory diagnosis of disseminated intravascular coagulation, deep vein thrombosis or pulmonary embolism. Most methods in use are either time-consuming, needing special techniques, or insensitive and poorly specific. Some time ago, Watanabe and Tullis described a simple and rapid, semiquantitative test to detect fm and fdp in plasma, based on the finding that ristocetin in low concentrations (1.0-1.5 mg/ml) can specifically precipitate fm and fdp. To 0.4 ml ACD plasma, 0.1 ml ristocetin (2.5 mg/ml) is added and vortexed. The mixture is then incubated for 30 min at 20°C and centrifuged at 50xg for 5 min. The test is considered to be positive when fibrin-like strands or small or large pellets are observed on the bottom of the tube. More recently, Pfliegler et al. reported that ristomycin (AGGRISTIN), a structural analogue of ristocetin, can replace ristocetin in this test.Here we report on further results with the ristomycin (AGGRISTIN) precipitation test in 138 patients with various intravascular thrombotic events. The results of this test, performed on ACD plasma, were compared to the serum fdp values detected by immunoelectrophoresis (IEF) and by the haemagglutina-tion inhibition test (HIT). In all 30 cases with serum fdp above 30 ug/ml (HIT) or 28 pg/ml (IEF), the precipitation test was positive; at lower fdp concentrations, as detected by HIT or IEF, the test still was positive in 70 per cent of these thrombosis patients, suggesting a superior sensitivity. In 16 patients with elevated fibrinogen levels (but no evidence of thrombosis), the test was positive in only 3. No false positive results were detected in 16 healthy controls. Preliminary results show that the minor disadvantage of the test (blood collection on acid citrate dextrose) may readily be overcome by the in vitro adjustment of the pH of citrate plasma, commonly used for other haemostatic tests, to between 7.0 and 7.4.On the basis of our results we suggest that the AGGRISTIN (ristomycin) precipitation test is a simple, rapid and reliable method for the laboratory diagnosis of intravascular clotting.

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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology

AJP Renal Physiology ◽

10.1152/ajprenal.00283.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. F1015-F1024 ◽

Cited By ~ 6

Author(s):

Ester M. Pereira ◽

Anatália Labilloy ◽

Megan L. Eshbach ◽

Ankita Roy ◽

Arohan R. Subramanya ◽

...

Keyword(s):

Fabry Disease ◽

Human Kidney ◽

Premature Death ◽

Antibody Array ◽

Cell Models ◽

Lysosomal Storage ◽

Protein Levels ◽

Gla Gene ◽

Fabry Nephropathy ◽

And Control

Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.

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Implementation of a Simplified Regional Citrate Anticoagulation Protocol for Post-Dilution Continuous Hemofiltration Using a Bicarbonate Buffered, Calcium Containing Replacement Solution

Blood Purification ◽

10.1159/000452755 ◽

2016 ◽

Vol 42 (4) ◽

pp. 349-355 ◽

Cited By ~ 6

Author(s):

Christopher J. Kirwan ◽

Ross Hutchison ◽

Sherif Ghabina ◽

Stephanie Schwarze ◽

Abigail Beane ◽

...

Keyword(s):

Metabolic Alkalosis ◽

Calcium Supplementation ◽

Regional Citrate Anticoagulation ◽

Prospective Audit ◽

Bleeding Events ◽

Continuous Hemofiltration ◽

Citrate Anticoagulation ◽

Replacement Solution ◽

Acid Citrate Dextrose ◽

Citrate Dextrose

Background/Aims: Recent updates to the Nikkiso Aquarius continuous renal replacement therapy (CRRT) platform allowed us to develop a post-dilution protocol for regional citrate anticoagulation (RCA) using standard bicarbonate buffered, calcium containing replacement solution with acid citrate dextrose formula-A as a citrate source. Our objective was to demonstrate that the protocol was safe and effective. Methods: Prospective audit of consecutive patients receiving RCA for CRRT within intensive care unit, who were either contraindicated to heparin or had poor filter lifespan (<12 h for 2 consecutive filters) on heparin. Results: We present the first 29 patients who used 98 filters. After excluding ‘non-clot' filter loss, 50% had a duration of >27 h. Calcium supplementation was required for 30 (30%) filter circuits, in 17 of 29 (58%) patients. One patient discontinued the treatment due to metabolic alkalosis, but there were no adverse bleeding events. Conclusion: Post-dilution RCA system is effective and simple to use on the Aquarius platform and results in comparable filter life for patients relatively contraindicated to heparin.

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Times to Key Events in the Course of Zika Infection and their Implications for Surveillance: A Systematic Review and Pooled Analysis

10.1101/041913 ◽

2016 ◽

Cited By ~ 9

Author(s):

Justin Lessler ◽

Cassandra T. Ott ◽

Andrea C. Carcelen ◽

Jacob M. Konikoff ◽

Joe Williamson ◽

...

Keyword(s):

Systematic Review ◽

Blood Supply ◽

Zika Virus ◽

Blood Donation ◽

Case Reports ◽

Fold Increase ◽

Pooled Analysis ◽

Control Measures ◽

History Of ◽

And Control

Background Evidence suggests that Zika virus has driven a 10-fold increase in babies born with microcephaly in Brazil, prompting the WHO to declare a Public Health Emergency of International Concern. However, little is known about the natural history of infection. These data are critical for implementation of surveillance and control measures such as protecting the blood supply. Methods We conducted a systematic review and pooled analysis to estimate the distribution of times from Zika infection to symptom onset, seroconversion, and viral clearance, and analyzed their implications for surveillance and blood supply safety. Results Based on 25 case reports, we estimate the median incubation period of Zika virus infection is 5.9 days (95% CI: 4.4-7.6), and that 95% of cases will develop symptoms by 11.1 days post-infection (95% CI: 7.6-18.0). On average seroconversion occurs 9.0 days (95% CI, 7.0-11.6) after infection, and virus is detectable in blood for 9.9 days (95% CI: 6.8-21.4). In 5% of cases detectable virus persists for over 18.9 days (95% CI: 12.6-79.5). The baseline (no screening) risk of a blood donation being infected with Zika increases by approximately 1 in 10,000 for every 1 per 100,000 person-days increase in Zika incidence. Symptom based screening reduces this by 7% (RR 0.93, 93% CI 0.86-0.99), and antibody screening by 29% (RR 0.71, 95% CI: 0.28-0.88). Conclusions Symptom or antibody-based surveillance can do little to reduce the risk of Zika contaminated blood donations. High incidence areas may consider PCR testing to identify lots safe for use in pregnant women.

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EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

Medical Immunology (Russia) ◽

10.15789/1563-0625-2019-1-137-140 ◽

2019 ◽

Vol 21 (1) ◽

pp. 137-140

Author(s):

O. V. Dolgikh ◽

N. V. Zaitseva ◽

D. G. Dianova ◽

A. V. Krivtsov ◽

K. D. Starkova ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Programmed Cell Death ◽

Annexin V ◽

Fold Increase ◽

Apoptosis Markers ◽

Stable Strontium ◽

Cell Death Mechanisms ◽

Apoptotic Pathways ◽

And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

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Regional anticoagulation with acid citrate dextrose-A for extracorporeal photoimmunochemotherapy

Vox Sanguinis ◽

10.1046/j.1423-0410.2002.00213.x ◽

2002 ◽

Vol 83 (3) ◽

pp. 222-226 ◽

Cited By ~ 7

Author(s):

R. Apsner ◽

B. Uenver ◽

G. Sunder-Plassmann ◽

R. M. Knobler

Keyword(s):

Acid Citrate Dextrose ◽

Citrate Dextrose ◽

Regional Anticoagulation

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Does Dingo Predation or Buffalo Competition Regulate Feral Pig Populations in the Australian Wet-Dry Tropics? An Experimental Study.

Wildlife Research ◽

10.1071/wr9950065 ◽

1995 ◽

Vol 22 (1) ◽

pp. 65 ◽

Cited By ~ 30

Author(s):

L Corbett

Keyword(s):

Experimental Study ◽

Sus Scrofa ◽

Interference Competition ◽

Fold Increase ◽

Bubalus Bubalis ◽

Northern Australia ◽

Dry Tropics ◽

Swamp Buffalo ◽

And Control ◽

The Relationship

Dingo (Canis farniliaris dingo) predation on feral pigs (Sus scrofa) in response to experimental changes in prey populations was measured over seven years in the seasonally wet-dry tropics of northern Australia. Following the removal of feral swamp buffalo (Bubalus bubalis) from half of the 614-km*2 study area, the number of pigs doubled and there was a 3-fold increase of pig in dingo diet. The relationship between the functional response of the dingo and pig abundance was negative and significant for both the treatment and control areas. This indicated that dingoes were not regulating the pig population. Instead, dingo predation probably acted in concert with interference competition by buffalo which decreased access to critical subterranean food for pigs during the dry season and thus limited population growth in pigs.

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Bisphenol A modulates the metabolic regulator oestrogen-related receptor-α in T-cells

Reproduction ◽

10.1530/rep-13-0423 ◽

2014 ◽

Vol 147 (4) ◽

pp. 419-426 ◽

Cited By ~ 18

Author(s):

Riccardo Cipelli ◽

Lorna Harries ◽

Katsuhiro Okuda ◽

Shin'ichi Yoshihara ◽

David Melzer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Bisphenol A ◽

Oxidative Metabolism ◽

Biological Effects ◽

Control Point ◽

White Blood Cells ◽

Fold Increase ◽

Metabolic Effects

Bisphenol A (BPA) is a widely used plastics constituent that has been associated with endocrine, immune and metabolic effects. Evidence for how BPA exerts significant biological effects at chronic low levels of exposure has remained elusive. In adult men, exposure to BPA has been associated with higher expression of two nuclear receptors, oestrogen receptor-β (ERβ) and oestrogen-related-receptor-α (ERRα), in peripheral white blood cells in vivo. In this study, we explore the expression of ESR2 (ERβ) and ESRRA (ERRα) in human leukaemic T-cell lymphoblasts (Jurkat cells) exposed to BPA in vitro. We show that exposure to BPA led to enhanced expression of ESRRA within 6 h of exposure (mean±s.e.m.: 1.43±0.08-fold increase compared with the control, P<0.05). After 72 h, expression of ESRRA remained significantly enhanced at concentrations of BPA ≥1 nM. Oxidative metabolism of BPA by rat liver S9 fractions yields the potent oestrogenic metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). Exposure of cells to 1–100 nM MBP increased the expression of both ESRRA (significantly induced, P<0.05, at 1, 10, 100 nM) and ESR2 (1.32±0.07-fold increase at 100 nM exposure, P<0.01). ERRα is a major control point for oxidative metabolism in many cell types, including T-cells. Following exposure to both BPA and MBP, we found that cells showed a decrease in cell proliferation rate. Taken together, these results confirm the bioactivity of BPA against putative T-cell targets in vitro at concentrations relevant to general human exposure.

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