Busadee Pratumvinit

Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.

The Effect of Vitamin D Status on Pediatric Asthma at a University Hospital, Thailand

Purpose In the USA and Europe, hypovitaminosis D is associated with increased asthma severity, emergency department (ED) visit, and impaired pulmonary function in asthmatic patients. However, in tropical countries, data on the effect of vitamin D status on asthma is limited. This study evaluates the relationship between vitamin D status and the level of asthma control as well as other asthmatic parameters. Methods Asthmatic children were evaluated for serum 25-hydroxyvitamin D, pulmonary function tests, a skin prick test, and the level of asthma control. Results A total of 125 asthmatic children were recruited (boys, 66.4%). Their mean age±SD was 10.8±3.0 years. Vitamin D statuses were: deficiency (<20 ng/mL) in 19.2% of the patients, insufficiency (20-30 ng/mL) in 44.8%, and sufficiency (>30 ng/mL) in 36%. The vitamin D levels were 25.9±9.4 ng/mL in uncontrolled patients, 29.2±8.6 ng/mL in partly controlled patients, and 27.9±8.0 ng/mL in controlled patients (P>0.05). There were no significant differences in pulmonary function, asthma exacerbation, inhaled-corticosteroid (ICS) dose, anti-inflammatory drugs, or ED visit or hospitalization between different vitamin D statuses. Vitamin D deficiency patients were older and had a delayed onset of asthma than insufficiency or sufficiency patients. There was no significant correlation between serum vitamin D and pulmonary function/doses of ICS. Conclusions High prevalences of vitamin D deficiency and insufficiency were found in asthmatic children in Thailand; however, there was no significant relationship between vitamin D status and the level of asthma control or other asthma parameters.

Validation and optimization of criteria for manual smear review following automated blood cell analysis in a large university hospital

CONTEXT Each laboratory should have criteria for manual smear review that limit workload without affecting patient care. The International Consensus Group for Hematology Review established guidelines for action after automated blood cell analysis in 2005. OBJECTIVE To compare the consensus group criteria with our laboratory criteria and optimize them for better efficiency. DESIGN A total of 2114 first-time samples were collected consecutively from daily workload and were used to compare 2 criteria as well as establish the optimized criteria. Another set of 891 samples was used to validate the optimized criteria. All samples were run on either Sysmex XE-5000 or Coulter LH750 hematology analyzers and were investigated by manual smear review. The efficiency of each set of criteria was compared and optimized to obtain better efficiency, an acceptable review rate, and a low false-negative rate. RESULTS From 2114 samples, 368 (17.40%) had positive smear results. Compared with that of our laboratory criteria, the efficiency of the consensus group criteria was higher (83.63% versus 78.86%, P < .001), the review rate was higher (29.33% versus 22.37%, P < .001), and the false-negative rate was lower (2.22% versus 8.09%, P < .001). After optimizing the rules, we obtained an efficiency of 87.13%, a review rate of 24.22%, and a false-negative rate of 2.98%. We validated the optimized criteria with another set of samples, and the efficiency, review rate, and false-negative rate were 87.32%, 25.25%, and 1.12%, respectively. CONCLUSIONS Each laboratory should verify the criteria for smear review, based on the International Consensus Group for Hematology Review, and optimize them to maximize efficiency.

Optimal criteria for microscopic review of urinalysis following use of automated urine analyzer.

BACKGROUND The Sysmex UX-2000 is a new, fully automated integrated urine analyzer. This device analyzes all physical and chemical characteristics of urine and sediments in urine on single platform. Because sediment analysis by fluorescent flow cytometry has limited ability to classify some formed elements present in urine (e.g., casts), laboratories should develop criteria for manual microscopic examination of urinalysis following the use of the automated urine analyzer. METHODS 399 urine samples were collected from routine workload. All samples were analyzed on the automated analyzer and were then compared to the results of the manual microscopic method to establish optimal criteria. Another set of 599 samples was then used to validate the optimized criteria. The efficiency of criteria and review rate were calculated. The false-positive and false-negative cases were enumerated and clarified. RESULTS We can set 11 rules which are related to the parameters categorized by the UX-2000, including cells, casts, crystals, organisms, sperm, and flags. After optimizing the rules, the review rate was 54.1% and the false-negative rate was 2.8%. CONCLUSIONS The combination of both UX-2000 and manual microscopic method obtain the best results. The UX-2000 improves efficiency by reducing the time and labor associated with the specimen analysis process.

Maternal Vitamin D Status and Its Related Factors in Pregnant Women in Bangkok, Thailand.

Background There are few data focusing on the prevalence of vitamin D deficiency in tropical countries. Objectives We determined the vitamin D status in pregnant women and examined the factors associated with vitamin D deficiency. Design and Methods A cross-sectional study of 147 pregnant Thai women aged 18–45 years at Siriraj Hospital (a university hospital in Bangkok, Thailand) was undertaken. Clinical data and plasma levels of 25-hydroxyvitamin D [25(OH)D], intact parathyroid hormone (iPTH), calcium, albumin, phosphate and magnesium were obtained in pregnant women at delivery. Results The prevalence of hypovitaminosis D [defined as 25(OH)D <75 nmol/L] in pregnant women at delivery was 75.5% (95% confidence interval (CI), 67.7–82.2%). Of these, vitamin D insufficiency [defined as 25(OH)D 50–74.9 nmol/L] was found in 41.5% (95% CI, 33.4–49.9%) and vitamin D deficiency [25(OH)D <50 nmol/L] was found in 34.0% (95% CI, 26.4–42.3%) of women. The mean 25(OH)D concentration was 61.6±19.3 nmol/L. The correlation between 25(OH)D and iPTH was weak (r = –0.29, P<0.01). Factors associated with vitamin D deficiency by multiple logistic regression were: pre-pregnancy body mass index (BMI in kg/m2, odds ratio (OR), 0.88, 95% CI 0.80–0.97, P = 0.01) and season of blood collection (winter vs. rainy, OR, 2.62, 95% CI 1.18–5.85, P = 0.02). Conclusions Vitamin D deficiency is common among pregnant Thai women. The prevalence of vitamin D deficiency increased in women who had a lower pre-pregnancy BMI and whose blood was collected in the winter. Vitamin D supplementation may need to be implemented as routine antenatal care.

Performance Evaluation and Comparison of the Fully Automated Urinalysis Analyzers UX-2000 and Cobas 6500

OBJECTIVE To evaluate and compare the performances of the automated urinalysis devices UX-2000 and Cobas 6500. METHOD A total of 258 urine specimens were collected from the routine specimen workload. We analyzed all specimens on both automated instruments and recorded the turnaround time from each method. Physical, chemical, and sedimentary urine components were compared between the automated and the manual method for each analyzer. RESULTS The correlation of urine physical/chemical properties between the 2 instruments was excellent. The Cobas 6500 instrument demonstrated a higher level of agreement for red blood cells (Cobas 6500:R= 0.94; UX-2000:R= 0.78) and white blood cells (Cobas 6500:R= 0.95; UX-2000:R= 0.85). The UX-2000 demonstrated higher sensitivity for small round cells, hyaline casts, pathological casts, and bacteria. The median turnaround time was 1.5 minutes and 8.5 minutes for the Cobas 6500 and UX-2000, respectively. CONCLUSIONS The 2 devices showed similar performance in technical evaluation; they each reduce workload and increase time saving. However, manual examination by technicians is recommended for pathological specimens.

Circulating free soluble fms-like tyrosine kinase-1 during late first trimester in relation with placental volume as a surrogate for trophoblastic production: a physiology study in low-risk cohort

Abstract Background: Data on first-trimester circulating soluble fms-like tyrosine kinase-1 (sFlt-1) and ischemic placental disease is limited and conflicting. This study aimed to study its physiology in relation to trophoblastic mass as the source of production. Methods: Low-risk (representing normal placentation) women from 11 0/7 to 13 6/7 weeks’ gestation were prospectively enrolled. Selective measurement of serum free sFlt-1 using a new automated assay from 100 eligible subjects was analyzed with gestational age, maternal weight, fetal crown-rump length (CRL), and mean uterine artery Doppler pulsatility index (PI). Placental volume (surrogate for trophoblastic mass) was estimated using 3-dimensional ultrasound and was assessed for its association with serum free sFlt-1. Results: There was no significant association between serum free sFlt-1 and placental volume in either arithmetic (r = 0.053, p = 0.600), logarithmic (r = 0.005, p = 0.963), or quartile (p = 0.703) scale. There was a significant negative correlation between free sFlt-1 level and maternal weight (r=−0.213, p = 0.033). No significant correlation was found between free sFlt-1 level and gestational age (r = 0.007, p = 0.947), CRL (r = 0.027, p = 0.788), and uterine artery Doppler mean PI (r = 0.020, p = 0.828). Conclusions: Lack of correlation between circulating free sFlt-1 level and placental volume suggests that trophoblasts are not its major source during first trimester with presumably physiologic placentation.

Circulating soluble fms-like tyrosine kinase-1 and placental growth factor from 10 to 40 weeks' pregnancy in normotensive women.

Abstract Introduction: Circulating soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) are potential markers for preeclampsia. The objective was to construct and analyse the reference ranges of serum levels of sFlt-1 and PlGF throughout the course of pregnancy in low-risk Thai pregnant women. Methods: We enrolled 110 low-risk, Thai women singleton pregnancy from 10 to 40 gestational weeks. Serum concentrations of sFlt-1 and PlGF were measured with an automated assay. The reference ranges of serum levels of sFlt-1, PlGF and sFlt-1/PlGF ratio were constructed and assessed for possible correlations with gestational age, maternal factors [age, parity, tobacco use, artificial reproductive technologies (ARTS) and body mass index (BMI)], and pregnancy outcomes (gestational age at delivery, development of preeclampsia, neonatal birth weight and placental weight). Results: None of the subjects developed preeclampsia. Serum sFlt-1 concentrations significantly elevated from 20 to 40 gestational weeks (P=0.003). Significant elevation and dropping of serum PlGF levels and sFlt-1/PlGF ratios were observed at 10 to 29 and 30 to 40 weeks of gestation, respectively (P<0.001). There was an inversed correlation between serum PlGF levels at 20 to 29 gestational weeks and neonatal birth weights (r=−0.48, P<0.05). There were no associations between serum levels of sFlt-1, PlGF, or sFlt-1/PlGF ratios and maternal BMI, gestational age at delivery, or placental weight (P>0.05). Effects from parity, smoking and ARTS were inconclusive. Conclusion: Robust change of serum PlGF levels suggests for its broader clinical application compared to sFlt-1. Prediction of preeclampsia using serum analytes may be gestational period specific.

The Effects of Temperature and Relative Humidity on Point-of-Care Glucose Measurements in Hospital Practice in a Tropical Clinical Setting.

Background: Hospitals in tropical countries experience conditions that exceed manufacturer temperature and humidity limits for point-of-care (POC) glucose reagents. Our goal was to assess the effects of out-of-limits storage temperature, operating temperature, and operating humidity on POC glucose measurement reliability. Methods: Quality control measurements were performed monthly using glucose test strips stored under controlled conditions and in inpatient wards under ambient conditions. Glucose test strips were evaluated in groups organized by operating temperatures of 24-25 (group 1), 28-29 (group 2), and 33-34°C (group 3), and relative humidity (RH) of ≤70 (group A), ~80 (group B), and ~90% (group C). Results: Glucose results for different storage conditions were inconsistent. Measurements at higher operating temperatures had lower values with mean differences of −2.4 (P < .001) and −36.5 (P < .001) mg/dL (28-29 vs 24-25°C), and −3.6 (P < .001) and −37.4 (P < .001) mg/dL (33-34 vs 24-25°C) for low and high control levels, respectively. Measurements at higher RH had lower values with mean differences of −4.0 (P < .001) and −13.2 (P < .001) mg/dL (~80 vs ≤70% RH), and −5.8 (P < .001) and −16.6 (P < .001) mg/dL (~90 vs ≤70% RH) for low and high levels, respectively. Conclusions: High temperature and high RH decreased glucose concentrations for the POC oxidase-based system we evaluated. We recommend that individual hospitals perform stress testing, then determine if maximum absolute differences, which represent highest risk for patients, are clinically significant for decision making by using error grid analysis.

Purification, Expansion and Characterization of Putative Murine CardiacProgenitor Cells

Ischemic heart disease kills more people than any other condition. Medical treatment for ischemic heart disease is currently limited by the heart’s lack of regeneration after injury. Recent reports have suggested the existence of cardiac progenitor cells in the adult normal and diseased mammalian heart. The origin of these cells is unclear. We implemented novel culturing conditions to isolate putative cardiac progenitor cells (pCPCs) from the adult murine heart atrium with the properties of cardiac regeneration. Putative adult cardiac progenitor cells were purified and expanded from adult murine hearts by expansion medium supplemented with 2% fetal calf serum, epidermal growth factor, platelet-derived growth factor and leukemia inhibitory factor. Under the culture condition of 5% O2 , these cells can be expanded beyond 42 days, expressed stem cell antigen (Sca-1) and cardiac-specific transcription factors. When treated with oxytocin in vitro, these cells express cardiac contractile proteins and when injected intramuscularly in the tibialis anterior muscle these cells give rise to cardiomyocyte-like cells. In contrast, transplantation of these pCPCs in uninjured heart did not result in cardiomyocyte differentiation suggesting that the heart environment is less permissive than the skeletal muscle of cardiogenesis. These results suggest that cells from the adult murine heart selected with this culture conditions and transplanted in the skeletal muscle have cardiogenic potential. Thus, this approach warrants further investigation for the therapeutic development of strategies for cardiac tissue engineering and myocardial regeneration.