Kanit Reesukumal

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.

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.

UriSed 3 and UX-2000 automated urine sediment analyzers vs manual microscopic method: A comparative performance analysis

Fully automated urine analyzers now play an important role in routine urinalysis in most laboratories. The recently introduced UriSed 3 has a new automated digital imaging urine sediment analyzer with a phase contrast feature. The aim of this study was to compare the performance of the UriSed 3 and UX‐2000 automated urine sediment analyzers with each other and with the results of the manual microscopic method.

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.

Should acidification of urine be performed before the analysis of calcium, phosphate and magnesium in the presence of crystals?

BACKGROUND Acidification of urine has been recommended before testing for calcium, phosphate, and magnesium. We investigated the necessity of pre-analytical acidification in both crystallized and non-crystallized urine samples. METHODS From 130 urine samples obtained via routine urine analysis, 65 (50%) samples were classified as non-crystallized. All samples were divided into three groups: untreated samples, acidified samples with HCl, and acidified samples after 1h room-temperature incubation. Urine samples were measured for calcium, phosphate, magnesium, and creatinine using Modular P800 and were examined for crystals using light microscopy. RESULTS In crystallized samples, acidified samples with 1h incubation had significantly higher Ca/Cr, P/Cr, and Mg/Cr than did untreated samples with mean differences of 0.04, 0.03, and 0.01 mg/mg, respectively (P<0.001). In acidified samples that were analyzed immediately, crystallized samples had lower calcium concentrations than those of acidified samples with 1h incubation and a mean difference of 0.21 mg/dl (P = 0.025). None of the sample differences which exceeded the critical difference of urinary Ca, P and Mg was observed. CONCLUSIONS Acidification of urine should be performed before the measurement of Ca, P, and Mg in the presence of urinary crystals. However, the lack of an acidification process does not result in a clinically significant change.