BACKGROUND: The identification of in vitro hemolysis (IVH) using a hematology analyzer is challenging because centrifugation of the specimens cannot be performed for cell counts. In the present study, we aimed to develop a scoring system to help identify the presence of hemolysis in anticoagulated blood specimens. METHODS: Thirty-seven potassium EDTA anticoagulated blood specimens were obtained, and each specimen was divided into 3 aliquots (A, B, and C). Aliquots B and C were mechanically hemolyzed by aspirating 2 and 5 times, respectively, using a 27-gauge needle and then tested; aliquot A was analyzed immediately without any hemolysis. After the cells were counted, aliquots B and C were centrifuged and the supernatants were tested for the hemolytic index and lactate dehydrogenase levels. RESULTS: The 4 hematologic parameters were selected and scored from 0 to 3 as follows:< 34.0, 34.0-36.2, 36.3-38.4, and > or =38.5 for mean cell hemoglobin concentration (MCHC, g/dL); <0.02, 0.02, 0.03, and > or =0.04 for red blood cell ghosts (10(12)/L); <0.13, 0.13-0.38, 0.39-1.30, and > or =1.31 for difference value (g/dL) of measured hemoglobin and calculated hemoglobin; and <0.26, 0.26-0.95, 0.96-3.34, and > or =3.35 for difference value (g/dL) of MCHC and cell hemoglobin concentration mean. The hemolysis score was calculated by adding all the scores from the 4 parameters. At the cutoff hemolysis score of 3, the IVH of aliquots B and C were detected as 64.9% and 91.9%, respectively. CONCLUSIONS: The scoring system might provide effective screening for detecting spurious IVH.
Anticoagulants/*pharmacology ; *Blood Specimen Collection ; Edetic Acid/pharmacology ; Hemoglobins/analysis ; Hemolysis/drug effects ; Humans

BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.
Bacterial Proteins/antagonists & inhibitors/*metabolism ; Boronic Acids/chemistry/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Edetic Acid/chemistry/pharmacology ; Enterobacteriaceae/drug effects/*enzymology ; Enterobacteriaceae Infections/diagnosis ; Humans ; Pseudomonas/drug effects/*enzymology ; Pseudomonas Infections/diagnosis ; Sensitivity and Specificity ; beta-Lactamases/chemistry/*metabolism

FOR smear layer removal from root canal walls, ethylenediaminetetraacetic acid (EDTA) is an effective chelating agent and its efficiency depends upon a lot of factors such as concentration, pH, duration of application, the type of the solution, the root canal length, penetration depth of the material, and hardness of the dentin.The aim of this scanning electron microscopic study was to evaluate the effectiveness of 19% EDTA gel on smear layer removal at different time periods when used as a final step in the irrigation regime.
Chelating Agents ; pharmacology ; Dental Pulp Cavity ; drug effects ; ultrastructure ; Edetic Acid ; pharmacology ; Gels ; Humans ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Root Canal Irrigants ; pharmacology ; Smear Layer ; Therapeutic Irrigation

This study is to investigate the effects of concentration, intestinal section, pH, paracellular route, substrate/inhibitor of enzyme (CYP3A) and proteins (P-gp, MRP2, SGL1) on the absorption of forsythoside A. The absorption of three concentrations (2.6, 5.2, and 10.4 microg x mL(-1)) of forsythoside A in different intestinal segments was studied with phenol red as the marker by rat circulation in situ. The results showed that the residue of forsythoside A with different concentrations had little significant difference from that obtained after perfusing via duodenum, jejunum, ileum and colon, which indicated that the absorption of forsythoside A was passive diffusion and had no difference in different segments of rat intestine. The residue of forsythoside A increased to 466.160 and 463.429 microg respectively when cyclosporine (4 microg x mL(-1)) or midazolam (50 micromol x L(-1)) was added to the circulation fluid, which showed significant difference compared to the control group (P < 0.05). Moreover, the residue of forsythoside A showed a tendency of increase with the increase of cyclosporine or midazolam. When digoxin (50 micromol x L(-1)) or EDTA (10 microg x mL(-1)) was added to the circulation fluid, the residue of forsythoside A decreased to 325.110 and 369.888 microg respectively, which showed significant difference as compared to the control group (P < 0.05). Besides, the residue of forsythoside A showed a tendency of reduction with the increase of digoxin or EDTA. However, there is no significant change in the absorption of forsythoside A when the different concentrations of mannitol were added to the circulation fluid. The results above indicated that the absorption of forsythoside A was mainly passive diffusion and involved paracellular route at the same time. In addition, the substrates of P-gp or CYP3A had dose-dependent effect on the absorption of forsythoside A.
Animals ; Colon ; metabolism ; Cyclosporine ; pharmacology ; Digoxin ; pharmacology ; Dose-Response Relationship, Drug ; Duodenum ; metabolism ; Edetic Acid ; pharmacology ; Glycosides ; administration & dosage ; pharmacokinetics ; Hydrogen-Ion Concentration ; Ileum ; metabolism ; Intestinal Absorption ; Jejunum ; metabolism ; Male ; Mannitol ; pharmacology ; Midazolam ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the different impacts of electrolytic iron, FeSO4, and NaFeEDTA on body iron store of anemic school students.

METHODSFour hundreds anemic students at the age of 11-18 years were divided into four groups. Of which, three consumed different iron fortificants from wheat flour as food vehicle for six months and one consumed non-fortified flour (control). The fortification level of electrolytic iron, FeSO4, and NaFeEDTA was 60 mg Fe/kg, 30 mg Fe/kg, and 20 mg Fe/kg, respectively. Blood samples were collected at 0, 2, 4, and 6 months and hemoglobin (Hb), serum ferritin (SF), and transferrin receptor (TfR) were measured.

RESULTSThe hemoglobin levels in three intervention groups increased, the increments of Hb in the NaFeEDTA group were significantly higher than that in the other groups. SF and TfR levels increased in the tested groups and body iron store in the NaFeEDTA group was higher than that in the other groups. These parameters did not show any significant changes in the control group.

CONCLUSIONNaFeEDTA and FeSO4 fortified wheat flour has positive impacts on iron status in anemic students and NaFeEDTA is more effective than FeSO4, while electrolytic iron is less effective in improving iron store in anemic students.

Adolescent ; Anemia, Iron-Deficiency ; drug therapy ; Child ; Dietary Supplements ; Dose-Response Relationship, Drug ; Edetic Acid ; chemistry ; pharmacology ; Female ; Ferric Compounds ; chemistry ; pharmacology ; Flour ; analysis ; Food, Fortified ; Humans ; Iron ; chemistry ; pharmacology ; Iron, Dietary ; Male ; Nutritional Status ; Triticum

BACKGROUND: The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples. METHODS: Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured. RESULTS: PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4degrees C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 micro mol/L leupeptin, 10 micro mol/L E-64, and 10 micro mol/L EDTA, respectively, in the samples stored at room temperature. CONCLUSIONS: The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail.
Aprotinin/pharmacology ; Blood Specimen Collection ; Edetic Acid/pharmacology ; Female ; Humans ; Leucine/analogs & derivatives/pharmacology ; Leupeptins/pharmacology ; Male ; Parathyroid Hormone/*blood/metabolism ; Protease Inhibitors/*pharmacology ; Time Factors

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.
Trypsin/pharmacology ; Stem Cells/*cytology/drug effects ; Rabbits ; Limbus Corneae/*cytology/drug effects ; Humans ; Epithelium, Corneal/*cytology/drug effects ; Edetic Acid/pharmacology ; Cells, Cultured ; Cell Culture Techniques ; Cell Count ; Animals

AIMTo explore the intestinal absorption characteristics of lumbrokinase (YJM-I) in the absence or presence of various absorption enhancers and to find the optimum intestinal site for YJM-I absorption.

METHODSThe absorption kinetics and absorption intestinal sites for YJM-I absorption were investigated with the method of diffusion cell in vitro, duodenum bolus injection, recirculating perfusion and in situ duodenum perfusion in vivo.

RESULTSYJM-I could be transported into blood and kept its biological activity across intestinal endothelial membrane after administration via duodenum site, whereas with lower bioavailability. Some of the absorption enhancers were shown good enhancement effects on intestinal absorption of YJM-I in vitro and in situ experiments. The order of enhanced efficiencies of various enhancers on duodenum, ileum and jejunum in vitro permeation experiments were shown as follows: 1% chitosan > 1% SDCh > 1% Na2EDTA > 1% SDS > 1% sodium caprylate > 1% poloxamer > 1% HP-beta-CD. The order of enhanced efficiencies of various enhancers on duodenum absorption of YJM-I in vivo were as follows: 2.5% SDCh > 2.5% Na2EDTA > 2.0% chitosan > 2.5% SDS > 2.5% sodium caprylate > 2.5% Poloxamer > 2.5% HP-beta-CD.

CONCLUSIONThe results indicated that the absorption of YJM-I could be enhanced by various enhancers, and duodenum was the optimum absorption site of YJM-I. Furthermore, bio-adhesive chitosan might be a potential enhancer of intestinal YJM-I absorption.

Administration, Oral ; Animals ; Area Under Curve ; Caprylates ; pharmacology ; Chitosan ; pharmacology ; Deoxycholic Acid ; pharmacology ; Duodenum ; drug effects ; metabolism ; Edetic Acid ; pharmacology ; Endopeptidases ; administration & dosage ; pharmacokinetics ; Injections, Intravenous ; Intestinal Absorption ; Male ; Metabolic Clearance Rate ; Poloxamer ; pharmacology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDWe assessed whether the CaNa2 EDTA could improve the accumulation of protoporphyrin IX (PpIX) and photosensitisation in HEp-2 cells as well as the depth of treatment of skin cancers on the topical 5-Aminolaevulinic acid (5-ALA) PDT.

METHODSHEp-2 cells were incubated with 5-ALA (0-1 mmol/L) and CaNa2EDTA (0-1 mmol/L) for 4 hours, intracellular protoporphyrin IX content was quantified by extraction, and cell viability was assessed by use of the methyl-tetrazolium (MTT) assay four hours after exposure to light. In comparison with the pictures before and after treatment, depth of treatment could be determined using a Acuson Sequioa 512 phase-array system in paired experiments.

RESULTSPpIX accumulation increased with increasing extracellular concentrations of ALA (0-1 mmol/L). Adding 1 mmol/L of CaNa2EDTA increased 30% PpIX accumulation over the same period of incubation in the concentration of 1 mmol/L ALA. Significant difference was observed between the 5-ALA alone group and 5-ALA combined CaNa2 EDTA group in the PpIX accumulation (P < 0.01). Cell viability after exposure to light decreased with adding CaNa2 EDTA, a statistical difference in a same fluence above 1.2 J/cm2 between two groups was demonstrated (P < 0.05, P < 0.01 respectively). Depth of treatment of skin cancers were increased in CaNa2 EDTA-treated group.

CONCLUSIONCaNa2 EDTA could improve the PpIX accumulation and photosensitisation in HEp-2 cells. Clinically, CaNa2 EDTA could increase the depth of treatment in the cutaneous cancers.

Aminolevulinic Acid ; therapeutic use ; Cell Line, Tumor ; Cell Survival ; Edetic Acid ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Photochemotherapy ; Protoporphyrins ; analysis ; Skin Neoplasms ; drug therapy

The study was purposed to explore the suitable platelet activators to be used in slide platelet aggregation test. Experiments were as follows: (1) to detect the intensity and time in 15 healthy donors' platelet aggregation tests induced by cationic propyl gallate (c-PG) and the usual platelet activators: ADP, collagen, epinephrine, arachidonic acid and ristocentin, respectively; (2) to detect the time in platelet aggregation tests of 15 healthy donors induced by c-PG and the above usual platelet activators respectively after addition of PGI2, cAMP or EDTA; (3) to detect the time in 15 healthy donors' platelet aggregation tests induced by c-PG after addition of heparin; (4) to detect the intensity and time of platelet aggregation induced by c-PG at the platelet count of (240-15) x 10(9)/L, (5) to detect the time of platelet aggregation induced by c-PG in eight patients each of whom had taken 100 mg aspirin per day for five days. The results showed that (1) c-PG reduced the strongest intensity of platelet aggregation and the time taken was appropriate, (2) c-PG was the most effective activator to reveal the inhibitive effect on platelet by PGI2, cAMP or EDTA, (3) 0.5 - 3 U/ml heparin did not significantly change the platelet aggregation induced by c-PG, (4) 15 healthy donors' platelet aggregation induced by c-PG displayed clearly on the slide until the platelet count below 30 x 10(9)/L, (5) The platelet aggregation time induced by c-PG was significantly prolonged in eight patients who had taken aspirin. In conclusion, compared to the usual platelet activators, c-PG has remarkable potential advantages when used in slide platelet aggregation test.
Cyclic AMP ; pharmacology ; Edetic Acid ; pharmacology ; Epoprostenol ; pharmacology ; Heparin ; pharmacology ; Humans ; Male ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Propyl Gallate ; pharmacology