A randomized cross-over study of cryopreserved platelets in prophylactic transfusions of thrombocytopenic patients.

BACKGROUND: The short shelf-life of liquid-stored platelets (LP) at 20-24°C poses shortage and wastage challenges. Cryopreserved platelets have significantly extended shelf-life, and were safe and efficacious for therapeutic transfusions of bleeding patients in the Afghanistan conflict and phase 2 randomized studies. Although hematology patients account for half of platelets demand, there is no randomized study on prophylactic cryopreserved platelet transfusions in them. METHODS: We performed a phase 1b/2a randomized cross-over study comparing the safety and efficacy of cryopreserved buffy coat-derived pooled platelets (CP) to LP in the prophylactic transfusions of thrombocytopenic hematology patients. RESULTS: A total of 18 adults were randomly assigned 1:1 to CP and LP for their first thrombocytopenic period (TP) of up to 28-days. A total of 14 crossed over to the other platelet-arm for the second TP. Overall, 17 subjects received 51 CP and 15 received 52 LP. CP-arm had more treatment emergent adverse event (29.4% vs. 13.3% of subjects, 9.8% vs. 3.8% of transfusions) than LP-arm but all were mild. No thromboembolism was observed. Both arms had similar bleeding rates (23.5% vs. 26.7% of subjects) which were all mild. Subjects in CP-arm had lower average corrected count increments than LP-arm (mean [SD] 5.6 [4.20] vs. 22.6 [9.68] ×109 /L at 1-4 h, p < .001; 5.3 [4.84] vs. 18.2 [9.52] ×109 /L at 18-30 h, p < .001). All TEG parameters at 1-4 h and maximum amplitude (MA) at 18-30 h improved from baseline post-CP transfusion (p < .05) though improvements in K-time and MA were lower than LP (p < .05). DISCUSSION: During shortages, CP may supplement LP in prophylactic transfusions of thrombocytopenic patients.

Changes in energy balance, body composition, metabolic profile and physical performance in a 62-day Army Ranger training in a hot-humid environment.

OBJECTIVES: To determine the physiological effects of multiple stressors including energy deficit during a 62-day Ranger course in a hot-humid environment. DESIGN: Prospective cohort design. METHODS: Food intake data were collected daily and energy expenditure at each of the three phases of the course was estimated by the doubly-labeled water method. Anthropometry, hydration status, stress and metabolic hormones, handgrip strength and lower explosive power were measured at the start and at the end of each phase. RESULTS: Seventeen male participants (age: 24.5 ±â€¯3.2 years, height: 173.9 ±â€¯5.1 cm, body mass: 69.3 ±â€¯3.2 kg, BMI: 22.9 ±â€¯0.9 kg/m2, percent body fat: 14 ±â€¯5%) completed the study. Mean total daily energy expenditure was 4756 kcal/day and mean daily energy intake was 3882 kcal/day. An 18% energy deficit resulted in an average body mass loss of 4.6 kg, comprising mostly fat mass. Participants with higher baseline adiposity (>15% body fat) lost more fat mass and gained (rather than lost) muscle mass compared to those with lower baseline adiposity. Handgrip strength declined only at the end of Phase I, while lower body explosive power declined progressively throughout the course. Lean mass in arms and legs was correlated with initial grip strength and lower body explosive power, but only at the start of the course. CONCLUSIONS: Physiologically demanding Ranger training in an equatorial environment is at least as metabolically demanding and stressful as other similar high-risk training courses, as demonstrated by the stress and metabolic endocrine responses, changes in body composition, and reduction in explosive power. Moreover, the smaller body size of Asian soldiers may confer an energetic advantage over larger sized Western counterparts.

Postprandial Glycemic and Insulinemic Effects of the Addition of Aqueous Extracts of Dried Corn Silk, Cumin Seed Powder or Tamarind Pulp, in Two Forms, Consumed with High Glycemic Index Rice.

Several plant-based traditional ingredients in Asia are anecdotally used for preventing and/or treating type 2 diabetes. We investigated three such widely consumed ingredients, namely corn silk (CS), cumin (CU), and tamarind (TA). The aim of the study was to determine the effects of aqueous extracts of these ingredients consumed either as a drink (D) with high-glycemic-index rice or added to the same amount of rice during cooking (R) on postprandial glycemia (PPG), insulinemia (PPI), and blood pressure (BP), over a 3 h measurement period. Eighteen healthy Chinese men (aged 37.5 ± 12.5 years, BMI 21.8 ± 1.67 kg/m2) took part in a randomized crossover trial, each completing up to nine sessions. Compared to the control meal (plain rice + plain water), the addition of test extracts in either form did not modulate PPG, PPI, or BP. However, the extracts when added within rice while cooking gave rise to significantly lower PPI than when consumed as a drink (p < 0.01). Therefore, the form of consumption of phytochemical-rich ingredients can differentially modulate glucose homeostasis. This study also highlights the need for undertaking randomized controlled clinical trials with traditional foods/components before claims are made on their specific health effects.

A Brugada syndrome proband with compound heterozygote SCN5A mutations identified from a Chinese family in Singapore.

AIMS: Brugada syndrome (BrS) is a rare heritable ventricular arrhythmia. Genetic defects in SCN5A, a gene that encodes the α-subunit of the sodium ion channel Nav1.5, are present in 15-30% of BrS cases. SCN5A remains by far, the highest yielding gene for BrS. We studied a young male who presented with syncope at age 11. This proband was screened for possible disease causing SCN5A mutations. The inheritance pattern was also examined amongst his first-degree family members. METHODS AND RESULTS: The proband had a baseline electrocardiogram that showed Type 2 BrS changes, which escalated to a characteristic Type I BrS pattern during a treadmill test before polymorphic ventricular tachycardia onset at a cycle length of 250 ms. Mutational analysis across all 29 exons in SCN5A of the proband and first-degree relatives of the family revealed that the proband inherited a compound heterozygote mutation in SCN5A, specifically p.A226V and p.R1629X from each parent. To further elucidate the functional changes arising through these mutations, patch-clamp electrophysiology was performed in TSA201 cells expressing the mutated SCN5A channels. The p.A226V mutation significantly reduced peak sodium current (INa) to 24% of wild type (WT) whereas the p.R1629X mutation abolished the current. To mimic the functional state in our proband, functional expression of the compound variants A226V + R1629X resulted in overall peak INa of only 13% of WT (P < 0.01). CONCLUSION: Our study is the first to report a SCN5A compound heterozygote in a Singaporean Chinese family. Only the proband carrying both mutations displayed the BrS phenotype, thus providing insights into the expression and penetrance of BrS in an Asian setting.

Evaluation of a prototype ration aimed at increasing caloric intake in a field environment.

Underconsumption is a common problem among militaries around the world. The purpose of this study was to review and modify the current field ration (FR) to encourage energy intake in the field. Military servicemen from the Singapore Armed forces (N = 232) were interviewed on their perception of the current FR. Based on their inputs, the FR was modified and subsequently evaluated by 434 military personnel undergoing field operations. Mean energy intakes of the servicemen measured through the daily collection of food wrappers were 1,651 ± 842 kcal/day (6.9 ± 3.5 MJ/day) and 1,237 ± 802 kcal/day (5.2 ± 3.4 MJ/day) for those on the modified (Test) and current (Control) FR, respectively. Surveys conducted at field sites indicated that servicemen in the Test group were less likely to skip meals and had higher desire to eat compared to those in the Control group. Factors that may have contributed to the higher energy intake include improved palatability and convenience, greater variety of food provided, and the higher nutrient density of the modified ration. The findings from this study have implications for future FR design.

Deep whole-genome sequencing of 100 southeast Asian Malays.

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.

Development of a next-generation sequencing method for BRCA mutation screening: a comparison between a high-throughput and a benchtop platform.

In a clinical setting, next-generation sequencing (NGS) approaches for the enrichment and resequencing of DNA targets may have limitations in throughput, cost, or accuracy. We evaluated an NGS workflow for targeted DNA sequencing for mutation detection. Targeted sequence data of the BRCA1 and BRCA2 genes, generated using a PCR-based, multiplexed NGS approach using the SOLiD 4 (n = 24) and Ion Torrent PGM (n = 20) next-generation sequencers, were evaluated against sequence data obtained by Sanger sequencing. The overall sensitivity for SOLiD and PGM were 97.8% (95% CI = 94.7 to 100.0) and 98.9% (95% CI = 96.8 to 100.0) respectively. The specificity for the SOLiD platform was high, at 100.0% (95% CI = 99.3 to 100.0). PGM correctly identified all 3 indels, but 68 false-positive indels were also called. Equimolar normalization of amplicons was not necessary for successful NGS. Both platforms are highly amenable to scale-up, potentially reducing the reagent cost for BRCA testing to

Rapid identification of chloroquine and atovaquone drug resistance in Plasmodium falciparum using high-resolution melt polymerase chain reaction.

BACKGROUND: Drug resistance determination for Plasmodium falciparum infections are important to determining the type of treatment to be given. Besides in vivo experiments, molecular methods, such as sequencing and PCR, are now increasingly being used. Here a cheaper alternative to sequencing or the use of multiplex 5'nuclease PCR assay for detection and differentiation of drug resistance haplotypes for chloroquine and atovaquone using polymerase chain reaction-high resolution melt (PCR-HRM) is reported. METHODS: Separate PCR-HRM assays were designed for the detection and differentiation of chloroquine and atovaquone drug resistance haplotypes in P. falciparum. PCR was conducted on a thermal cycler and melt curves generated using a LightScanner. These were tested against reference strains of P. falciparum from MR4 as well as 53 local isolates. RESULTS: The PCR-HRM assays are able to detect and differentiate between the various haplotypes consistently. These assays can also be used to detect new variants. CONCLUSIONS: PCR-HRM is an inexpensive option for the determination of drug resistance profile in P. falciparum and will see increasing use as an alternative to sequencing and 5'nuclease PCR assays in reference laboratories or once PCR systems that are able to conduct HRM become commonplace.

Polymorphism studies of six miniSTR loci for three ethnic populations in Singapore.

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimen, because of the improved PCR efficiency of their reduced PCR amplicon sizes. This study constructed two miniSTR panels comprising six miniSTR loci, including D2S1776, D3S4529, D6S474, D9S2157, D10S1435 and D12ATA63. The allele frequency distribution, forensic parameters and heterozygosity in three Singapore populations are reported in this paper. All six loci showed relatively high polymorphism with observed heterozygosity > 0.7. However, many of these six loci also demonstrated significant population differentiation for the three populations.

Distribution of PON1 polymorphisms PON1Q192R and PON1L55M among Chinese, Malay and Indian males in Singapore and possible susceptibility to organophosphate exposure.

Organophosphate (OP)-containing pesticides are widely used worldwide for domestic and industrial purposes. Studies on acute and chronic exposure to OPs have revealed numerous health effects attributed mainly to acetylcholinesterase (AChE) inhibition. The enzyme human serum paraoxonase (PON1) is involved in the detoxification of OP compounds. PON1 polymorphisms have been shown to affect susceptibility to OP exposure. We studied the effect of OP exposure on pest control workers and assessed the distribution of two common PON1 polymorphisms in our local population. The exposed group consisted of 103 workers from various pest control companies under the Singapore Pest Management Association while the 91 unexposed workers were from a lead stabilizer factory. For all workers, the mean age was 36.9 (20-70) years and the ethnic distribution was 38.1% Chinese, 44.3% Malay and 17.5% Indian. The mean+/-S.D. exposure duration among the pesticide workers was 10.4+/-8.4 years. The mean+/-S.D. RBC cholinesterase level was 18436.2+/-2078U/L and 18079.6+/-1576U/L for the exposed and unexposed groups, respectively (p=0.216). The mean+/-S.D. serum pseudocholinesterase was 11028.4+/-2867.4U/L and 9433.6+/-2022.6U/L in the exposed and unexposed groups, respectively (p<0.0001). Mean paraoxonase activity was similar among Chinese and Malays (266.5 and 266.3U/L, respectively) whereas that of the Indians was significantly lower (165.6U/L). Our study showed that cholinesterase levels among the exposed were not lower than those in the unexposed group. PON1 polymorphisms differed among ethnic groups, implying that ethnicity could be an important surrogate for identifying susceptible groups in case of OP exposure. Although OP poisoning is rare among occupationally exposed workers in Singapore, this information is useful for other developing countries that have large populations of Chinese, Malays and Indians where OP exposure could be very high especially in agricultural settings.

Molecular characterization of a polymorphic 3-Mb deletion at chromosome Yp11.2 containing the AMELY locus in Singapore and Malaysia populations.

Amelogenin paralogs on Chromosome X (AMELX) and Y (AMELY) are commonly used sexing markers. Interstitial deletion of Yp involving the AMELY locus has previously been reported. The combined frequency of the AMELY null allele in Singapore and Malaysia populations is 2.7%, 0.6% in Indian and Malay ethnic groups respectively. It is absent among 541 Chinese screened. The null allele in this study belongs to 3 Y haplogroups; J2e1 (85.7%), F* (9.5%) and D* (4.8%). Low and high-resolution STS mapping, followed by sequence analysis of breakpoint junction confirmed a large deletion of 3 to 3.7-Mb located at the Yp11.2 region. Both breakpoints were located in TSPY repeat arrays, suggesting a non-allelic homologous recombination (NAHR) mechanism of deletion. All regional null samples shared identical breakpoint sequences according to their haplogroup affiliation, providing molecular evidence of a common ancestry origin for each haplogroup, and at least 3 independent deletion events recurred in history. The estimated ages based on Y-SNP and STR analysis were approximately 13.5 +/- 3.1 kyears and approximately 0.9 +/- 0.9 kyears for the J2e1 and F* mutations, respectively. A novel polymorphism G > A at Y-GATA-H4 locus in complete linkage disequilibrium with J2e1 null mutations is a more recent event. This work re-emphasizes the need to include other sexing markers for gender determination in certain regional populations. The frequency difference among global populations suggests it constitutes another structural variation locus of human chromosome Y. The breakpoint sequences provide further information to a better understanding of the NAHR mechanism and DNA rearrangements due to higher order genomic architecture.

Polymorphism studies of four miniSTR loci for three ethnic populations in Singapore.

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR amplicon sizes. This study constructed a partial miniSGM panel and investigated the performance of four miniSTR loci, D2S1338, D16S539, D18S51 and FGA, in three ethnic populations residing in Singapore. The suitability of the miniSTR primers for Singapore populations was assessed for loci D16S539, D18S51 and FGA.

Heterozygosities and allelic frequencies of a set of microsatellite markers used for genome-wide scans in a Chinese population.

Microsatellite (short tandem repeat) markers are useful tools for genetic linkage analysis because of their high frequency of occurrence in eukaryotic genomes, ease of typing, and high polymorphism content. To establish a panel of microsatellite markers useful for genome-wide screens in the Chinese population, we determined the heterozygosities and allelic frequencies of a widely used set of 285 markers in 208 individuals of Han Chinese descent. Although the median heterozygosity level in our Chinese population was 0.72, only 63.6% of these markers have a heterozygosity of at least 0.7, compared with 90.8% in the original Caucasian sample. The significant difference in heterozygosity and allelic frequencies between populations suggests that markers should be optimally selected for each study population to maximize information content and power.