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ORIGINAL ARTICLE. Supporting information may be found in the online version of this article.
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1 DOI: /pd.4002 ORIGINAL ARTICLE Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in pregnancies with mixed risk factors Shan Dan 1,2,WeiWang 1, Jinghui Ren 3,YaliLi 4,HuaHu 5, Zhengfeng Xu 6,TzeKinLau 7,JianhongXie 8,WeihuaZhao 9,HefengHuang 10,11, Jiansheng Xie 12,LumingSun 13, Xiaohong Zhang 14,WeipengWang 15, Shixiu Liao 16,RongQiang 17,JiangxiaCao 18, Qiufang Zhang 19, Yulin Zhou 20, Haiyan Zhu 21, Mei Zhong 22,YiGuo 23, Linhua Lin 3, Zhiying Gao 4,HongYao 5, Hongyun Zhang 1, Lijian Zhao 1,FumanJiang 1, Fang Chen 1,HuiJiang 1, Songgang Li 1,YingruiLi 1,JunWang 1,JianWang 1, Tao Duan 13 *,YueSu 2 * and Xiuqing Zhang 1 * 1 BGI-Shenzhen, Shenzhen, China 2 Department of Perinatology, Beijing Obstetrics and Gynecology Hospital, Capital University of Medical Sciences, Beijing, China 3 The Center of Prenatal Diagnosis, Shenzhen People s Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, China 4 Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing, China 5 Southwest Hospital, The Third Military Medical University, Chongqing, China 6 Center of Prenatal Diagnosis, Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing, China 7 Fetal Medicine Centre, Paramount Clinic, Hong Kong 8 Zhuhai Institute of Medical Genetics, Zhuhai Municipal Maternal and Child Healthcare Hospital, Zhuhai, China 9 Department of Obstetrics, The Second People s Hospital of Shenzhen, The First Affiliated Hospital of Shenzhen University, Shenzhen, China 10 Key Laboratory of Reproductive Genetics, Zhejiang University, Ministry of Education, Hangzhou, China 11 Department of Reproductive Endocrinology, Women s Hospital, Zhejiang University School of Medicine, Hangzhou, China 12 Central for Prenatal Diagnosis, Shenzhen Maternity and Child Healthcare Hospital, Affiliated Southern Medical University, Shenzhen, China 13 Department of Obstetrics, Shanghai 1st Maternity and Infant Hospital, Tongji University, Shanghai, China 14 Department of Obstetrics and Gynecology, Peking University People s Hospital, Beijing, China 15 Department of Clinical Laboratory, Hubei Maternal and Child Health Hospital, Wuhan, China 16 Medical Genetic Center, Henan Provincial People s Hospital, Zhengzhou University People s Hospital, Zhengzhou, China 17 Prenatal Diagnosis Center, Maternal and Child Health Hospital of Shanxi Province, Xi an, China 18 Department of Obstetrics and Gynecology, Wuhan Medical Care Center for Women and Children, Wuhan, China 19 Department of Perinatal Medicine, Peking University Third Hospital, Beijing, China 20 Xiamen Prenatal Diagnosis Center, Xiamen Maternal and Child Health Care Hospital, Xiamen, China 21 Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China 22 Department of Gynecology and Obstetrics, Nanfang Hospital, First Military Medical University, Guangzhou, China 23 Department of Perinatal Medicine, Dalian Obstetrics and Gynecology Hospital, Dalian, China *Correspondence to: Xiuqing Zhang. or Yue Su. or Tao Duan. These authors contributed equally to this work. ABSTRACT Objective To report the performance of massively parallel sequencing (MPS) based prenatal noninvasive fetal trisomy test based on cell-free DNA sequencing from maternal plasma in a routine clinical setting in China. Method The MPS-based test was offered as a prenatal screening test for trisomies 21 and 18 to pregnant women in 49 medical centers over 2 years. A total of participants were recruited and the MPS-based test was performed in pregnancies. Fetal outcome data were obtained after the expected date of confinement. Results One hundred ninety cases were classified as positive, including 143 cases of trisomy 21 and 47 cases of trisomy 18. With the karyotyping results and the feedback of fetal outcome data,we observed one false positive case of trisomy 21,one false positive case of trisomy 18 and no false negative cases, indicating 100% sensitivity and 99.96% specificity for the detection of trisomies 21 and 18. Conclusion Our large-scale multicenter study proved that the MPS-based test is of high sensitivity and specificity in detecting fetal trisomies 21 and 18. The introduction of this screening test into a routine clinical setting could avoid about 98% of invasive prenatal diagnostic procedures. Supporting information may be found in the online version of this article.
2 S. Dan et al. Funding sources: The study was funded by Shenzhen Birth Defect Screening Project Lab (JZF No. [2011] 861) approved by Shenzhen Municipal Commission for Development and Reform and Key Laboratory Project in Shenzhen (CXB A and CXB A) and Key Laboratory of Cooperation Project in Guangdong Province (2011A ). Conflicts of interest: Wei Wang, Hongyun Zhang, Lijian Zhao, Fuman Jiang, Fang Chen, Hui Jiang, Songgang Li, Yingrui Li, Jun Wang, Jian Wang, Xiuqing Zhang are employees of BGI-Shenzhen and none of the other authors have any financial relationship with BGI-Shenzhen. INTRODUCTION Each year there are about 16 million newborns in China, of whom 4% to 6% are affected by some form of birth defects. 1,2 Chromosomal abnormalities, with an incidence of 1 in 160 births, are one of the most important causes of birth defects, and there are no curative treatments at present. 3 Although invasive prenatal tests allow accurate diagnosis, wide spread clinical use is limited by cost and a 0.5% to 1% risk of procedure-related miscarriage. 4 6 Over the last three decades, many screening tests have been developed to identify the highrisk groups for the most common chromosomal abnormalities, such as trisomy 21 (T21), 18 (T18) and 13 (T13), by various combinations of medical history, maternal age, ultrasound markers and maternal serum biochemistry. To date the first trimester combined screening has become widely accepted, being able to detect about 90% of trisomy 21 fetuses and 50% to 85% of other chromosomal defects. 7,8 However, at a false positive rate of 4% to 5% for current screening approaches, only about 5% of the high-risk pregnant women indeed carry a fetus with trisomy 21 while the remaining are normal, giving a positive predictive value of about 1 in 20. 9,10 Therefore, there is a continuous drive to search for a diagnostic test without risk of miscarriage, or a screening test with better performance. A variety of strategies have been explored using maternal blood, urine, and saliva samples to diagnose fetal gender and trisomy ,12 The discovery of the presence of fetal cell-free DNA and RNA in maternal plasma, combined with PCR technologies and mass spectrometric analysis, has enabled noninvasive detection of trisomies 21 and 18 with acceptable sensitivity and specificity However, these methods mainly detect specific alleles in a certain population, which has restricted its wide and robust application in clinical practice. In 2008 the rapid development of massively parallel sequencing (MPS) technology made it feasible to use maternal plasma cellfree DNA to detect trisomy 21 without using polymorphic markers. 18,19 Studies from multiple centers so far have shown that this is a highly reliable approach with a detection rate of 99% to 100% and a specificity of 98% to 100% for trisomy More recent studies have suggested that, with improved bioinformatic analysis, similar accuracies are achievable for the detection of trisomies 18 and Several research groups have published their clinical trial data with 397 to 4664 cases. 22,23,27,28 Here we reported the results of a prospective large-scale multicenter study in a routine clinical setting in a Chinese population in which participants were recruited and took the MPS-based test. Our experience may provide useful information to clinicians and medical practitioners who are interested in the application and integration of this new technology with established prenatal screening and diagnosis workflow. METHODS Participants and overall design In a 2-year period from the first quarter of 2010 to the first quarter of 2012, the MPS-based test was offered to pregnant women as a clinical screening test in 49 medical centers in 15 provinces and 4 centrally administered municipalities, all of which are prenatal screening or diagnosis centers certified by the Ministry of Health (MOH) of China. The inclusion criteria for participants were: (1) pregnant women, at least 18 years old or above, (2) with a singleton live fetus and (3) a gestational age of 9 to 28 weeks. Women with multiple pregnancies or intra-uterine fetal demise at the time of sampling or unknown gestational age were excluded from this study. Cases with an expected date of confinement before 31th March 2012 or those with karyotyping results were included for the current analysis. Approvals were obtained from the institutional review board of BGI-Shenzhen. Informed written consent was obtained from all pregnant women who agreed to take this screening test. All MPS-based tests were performed prior to the recording of karyotyping information and the sequencing lab in Shenzhen was blinded. Each collaborating site was responsible for obtaining fetal outcome data for their own study subjects. To minimize under-reporting of false negative cases among the negative cases, and to motivate participants to report undiagnosed trisomies 21 and 18, we have established an insurance scheme for this clinical study, as described below. THE MPS-BASED TEST Pretest counseling Careful pretest counseling was provided to each participant. All participants were informed of the limitations of the MPSbased test as follows: (1) This test is mostly intended for the prenatal detection of fetal trisomies 21 and 18 at the gestational age of 12 to 24 weeks. (2) Although the reported sensitivities and specificities for trisomies 21 and 18 were as high as 99% or above, the MPS-based test is still a screening test. All test positive pregnancies should receive confirmatory invasive testing. (3) About 1% of pregnancies would need resampling because of failure in the quality control criteria. (4) This test cannot be applied to the following situations: (a) when the pregnant woman is affected with trisomy 21 or 18, (b) at an earlier gestational age than 9 weeks, or (c) women with multiple pregnancies.
3 Clinical application of sequencing-based prenatal noninvasive fetal trisomy test (5) This test is not designed to identify fetuses with mosaicism, triploidy or chromosomal microdeletions/microduplications. Besides the information described above, on the consent form all participants were informed that they automatically joined the insurance scheme purchased by BGI-Shenzhen on their behalf, if they accept our test. In the event that a pregnant woman had a test negative result but eventually delivered a baby with trisomy 21 or 18, the insurance company would pay her CNY (equal to $31 658) (China Life Insurance Company, Ltd.). Massively parallel DNA sequencing Five milliliters of peripheral venous blood sample were collected into tubes containing Ethylene Diamine Tetraacetic Acid from each participant. All blood samples were obtained before any invasive prenatal diagnostic test, and were processed within 8 h by a double-centrifugation protocol. Blood samples were first centrifuged at 1600g for 10 min, and the supernatant was recentrifugated at g for 10 min to remove residual cells. The resultant cell-free plasma was stored at 80 C, and then shipped to BGI-Shenzhen on dry ice. Most samples arrived at the lab within 24 h and very few arrived no more than 72 h. Each plasma sample was frozen and thawed only once. For DNA extraction, 600 ml of maternal plasma was used. The DNA libraries were prepared according to a modified protocol from Illumina. After end-repairing, A-base tailing and adaptor ligation, standard multiplex primers were introduced by 17-cycle PCR. The size distribution of the libraries was analyzed using an Agilent Bioanalyzer and quantified with real-time PCR. Four or 12 barcoded libraries were equally pooled and sequenced with 36-cycles single-end multiplex sequencing strategy on an Illumina GAIIx or HiSeq 2000 platform, respectively. All molecular tests and procedures were performed in an ISO/IEC certified and MOH accredited clinical laboratory at BGI-Shenzhen. Quality control in sampling and sequencing We set up a quality control criteria system for each step from sampling to reporting. A barcode tracking system was employed during the whole process. Blood samples with evidence of hemolysis or those that were processed beyond 8 h after sample collection were excluded. The quality parameters of acceptable samples were as follows: the peak size of a qualified DNA library was between 290 and 303 bp, and the yield more than 30 nm. The sequencing quality value (Q20) was over 90% for each base, and the GC content was around %. The minimal amount of unique sequencing reads was no less than 2 million after alignment, which corresponded to about 5 million raw sequencing reads. Only qualified sequencing data were used for subsequent analyses. Trisomies 21 and 18 analysis Thirty-five base sequencing reads were trimmed and aligned back to a universal unique read set incised from the human reference genome (HG 18, NCBI build 36). A binary hypothesis t-test and logarithmic likelihood ratio L-score between the two t-tests were used to classify whether the fetus had trisomy 21 or 18 or not (a detailed description of the methodology is available in supplementary materials). If both the t-score >2.5 and the L-score >1, the sample was located in the high risk zone. If either the t-score >2.5 or the L-score >1 the sample was located in the warning zones. If the t-score <2.5 and the L-score <1, samples were located in the low-risk zone. Samples located in the high risk zone or warning zones were classified as test positive, while samples located in low risk zone as test negative. Data and statistical analysis The sensitivity and specificity of the MPS-based test was evaluated, using the result of fetal karyotyping as the gold standard. 95% confidence intervals (CIs) were calculated on the bases of a standard normal distribution. Report delivery and posttest counseling The report of our test would be issued within 15 working days from blood drawing. Posttest counseling was provided by clinicians and careful advice was given according to the test results. Follow up investigation In the case of MPS-based test positive results, participants were advised to have prenatal fetal karyotyping. If refused, the outcome of the case was monitored intensively to ensure a complete follow-up of the pregnancy. In the case of MPS-based test negative results, the decisions for prenatal fetal karyotyping were made according to other clinical parameters and the wish of individual participants. Fetal outcome data were obtained by questionnaires from participants. Figure 1 The workflow of the MPS-based test. The MPS-based test was offered as a screening test with careful pre-test and posttest counseling. The test was performed in an ISO/IEC17025 certified and MOH accredited clinical laboratory, where a laboratory information management system was employed throughout the entire workflow of activities to guarantee end-to-end traceability. In this study, test positive cases were referred for conventional invasive procedures while negative cases for intensive monitoring to ensure a complete follow-up data
4 S. Dan et al. RESULTS Workflow Figure 1 summarizes the workflow of the MPS-based test. In general, 95% of cases received the report within 10 working days from blood sampling, 99% of our test reports were issued within 15 working days. Resampling of pregnancies During the study, 0.87% (97/11 105) pregnancies needed resampling because of failure in quality control criteria mostly because of early gestational age. The test for these cases usually took another 10 to 15 working days from resampling to report delivery. receive results because of recruitment criteria failure or quality control failure. Basic characteristics of the study population are shown in Table 1. The maternal age ranged from 18 to 49 years old with a median of 31. The median gestational age at the time of blood sampling was 20 weeks % (3858/11 105) pregnancies were 35 years old or above % (6948/11 105) received either first or second trimester conventional Down syndrome screening tests, in which 65.08% (4522/6948) were screen positive. 2.85% (317/11 105) had ultrasound markers of chromosomal abnormalities, and 0.61% (68/11 105) had a family history of chromosomal abnormalities. No specific risk factor for chromosomal abnormalities was present in 12.49% of the tested cases (1387/11 105). Study participants Forty-nine medical centers participated in this clinical study, and a total of pregnant women were recruited in two years (Figure 2). Forty-two participating centers offered the test to high risk pregnant women identified by a conventional Down syndrome screening test, and the remaining 7 centers enrolled participants regardless of prior risk assessment. The MPS-based test was not performed in 79 cases because the recruitment criteria were not met. Another 79 cases failed in the quality control in the plasma separation, DNA extraction, sample preparation or sequencing steps mentioned in the methods section. Overall, our test was performed on the remaining cases. Also, 1.5% (158/11 263) of the women did not Identification of trisomies 21 and 18 by the MPS-based test An average of 5 million sequencing reads per sample was obtained using the Illumina GAIIx or HiSeq 2000 sequencing platforms. After removal of low quality reads and alignment, at least 2 million unique reads for each sample were used for the detection of trisomies 21 and 18. One hundred ninety cases were classified as test positive, giving a test positive rate of 1.71% (95% CI, 1.47% 1.95%). Of those, 143 cases were positive for trisomy 21 and 47 cases for trisomy 18, corresponding to an incidence of 1.29% (95% CI, 1.08% 1.50%) for trisomy 21, and 0.42% (95% CI, 0.30% 0.54%) for trisomy 18 in this cohort (Figure 3). Figure 2 Participants recruitment and overall design. A total of pregnant women were recruited in this multicenter study. Seventy-nine cases failed to meet the recruitment criteria and another 79 cases failed in the quality control. The MPS-based test was performed on the remaining cases, among which 3000 cases had performed full karyotyping and 4524 pregnancies had follow-up fetal outcome data
5 Clinical application of sequencing-based prenatal noninvasive fetal trisomy test Table 1 Basic characteristics of the pregnant women who took the MPS-based test Maternal age Median (yr) 31 Advanced maternal age ( 35 yr) 3858(34.74%) yr (n, %) 918(8.27%) yr (n, %) 3439(30.97%) yr (n, %) 2890(26.02%) yr (n, %) 3188(28.71%) 40 yr (n, %) 670(6.03%) Parity Nullipara (n) 6053/11 105(54.51%) Gestational age at blood sampling Median (wk) 20 Range (wk) 9 28 Follow-up investigation of test positive cases One hundred forty of 143 T21-positive cases were confirmed by prenatal karyotyping, including 138 cases of typical trisomy 21, one case of mosaic trisomy 21 (23 of 50 cells studied were trisomic), and one case with euploid karyotype. Three test positive cases did not have karyotype analysis because of spontaneous abortion, intra-uterine fetal demise, or induced abortion, respectively. Forty-two of 47 T18-positive cases were validated by prenatal karyotyping, and 41 cases were confirmed to be typical trisomy 18 and one case with euploid karyotype. Karyotyping was not performed in five test positive cases, including four cases of induced abortion, and one case of intra-uterine infection. Among these five cases, one had nuchal translucency >6 mm, one case had abnormal ultrasound markers and the other three cases were at high risk of trisomy 18 based on maternal biochemical screening. 9 to 12 weeks (n, %) 371(3.34%) 13 to 16 weeks (n, %) 1963(17.68%) 17 to 20 weeks (n, %) 5598(50.41%) 21 to 24 weeks (n, %) 2665(24.00%) 25 to 28 weeks (n, %) 508(4.57%) History or family history of aneuploidies (n) 68/11 105(0.61%) Presence of sonographic markers of chr 317/11 105(2.85%) abnormalities (n) Had conventional Down syndrome screening tests Yes Screening positive (n, %) 4522/6948(65.08%) Yes Screening negative (n, %) 2426/6948(34.92%) No with one or more of other risk factors a (n, %) 2770/11 105(24.94%) No without any risk factors a (n, %) 1387/11 105(12.48%) a Other risk factors refer to advanced maternal age, presence sonographic markers of a positive history of affected pregnancies. Follow-up investigation of test negative cases Overall, cases were classified as test negative for both trisomies 21 and 18. Among them, 2818 also had fetal karyotyping performed. The indications for the invasive procedures were: (1) positive conventional Down syndrome screening tests in 1178 subjects, (2) abnormal sonographic findings in 56 cases, (3) advanced maternal age in 757 cases, and (4) other reasons such as maternal anxiety or family history in 827 cases. Trisomy 21 or 18 was not detected in any of these 2818 karyotyping studies. In the remaining 8097 test negative cases without karyotyping, fetal outcome data and detailed information from the newborn examination was available for 4524 pregnancies, which showed that no woman gave birth to a trisomy 21 or 18 newborn. So far, no claim has been received for the insurance coverage for the birth of an affected newborn missed by the MPS-based test. Figure 3 Identification of fetal aneuploidy by the MPS-based test. The risk of fetal aneuploidy was described by the L-score (x-axis) and t-score (y-axis). Thetrueaneuploidyandnegativesamplebyfetalkaryotpyingislabeledbyasolidtriangle( ) and hollow circles ( ). The high-risk zone is defined by L-score > 1andt-score > 2.5. (A) 143 cases (including one T21 false positive case) were located in the high-risk zone for trisomy 21 and classified as T21-positive. (B) 47 cases (including one T18 false positive case) were located in the high-risk zone for trisomy 18 and classified as T18-positive
6 S. Dan et al. The estimation of sensitivity and specificity of the MPS-based test Overall, 3000 cases had full karyotyping performed. There were 139 cases of trisomy 21, 41 cases of trisomy 18, and 2820 euploid cases. We observed one false positive case of trisomy 21 and one false positive case of trisomy 18 in our test. Therefore, the detection rate of the MPS-based test in identifying fetal trisomy 21 and 18 were 100% (139/139) and 100% (41/41), whereas the false positive rate were 0.03% (1/2820) and 0.03% (1/2820), respectively. The estimated sensitivities and specificities were 100% and 99.96% for both trisomies 21 and 18, respectively. DISCUSSION In this study pregnant women from 49 medical centers were recruited in China, to evaluate the performance of the MPS-based test as a screening test in a clinical setting. This multicenter study recruited a relatively large sample size involving participants with various clinical risk factors. It was performed in the routine and complicated clinical settings. With proper pretest and posttest counseling, our test results were shown to be highly accurate, helping clinicians to offer better medical advices to pregnant women who then can make well-informed pregnancy decisions. The detection rate (or sensitivity) and the false positive rate (or specificity) are critical parameters in evaluating a new clinical testing technology. In this study, 182 of 190 test positive cases had fetal karyotyping performed. Our test successfully identified all true trisomies 21 and 18 cases; however, there were two false positive cases. For those eight cases identified as test positive but did not have karyotyping, we believe it was unlikely to be false positive, because these cases showed high-risk indications in conventional screening tests and ended with abnormal pregnancy outcomes, such as spontaneous abortion, and intrauterine fetal demise. Taken together, the estimated specificity of 100% for trisomies 21 and 18 was likely to be reasonable. Even if all these eight cases were false positive, the false positive rate would still be very low, less than 1/1000. Among the test negative cases, 2818 (25.82%) women still requested prenatal fetal karyotyping results. This was probably because many pregnant women were still uncertain about the reliability of our test at that moment, and needed reassurance from conventional invasive tests. On the other hand, this gave us the opportunity to exclude false negativity of our test with certainty among these cases, because the karyotyping results were all normal for both chromosome 21 and 18. Six hundred twenty-five of 2818 cases were still pregnant at the time this manuscript was written. However, follow-up investigation of the remaining 8097 test negative cases without karyotyping was a challenge. In China, follow-up data are difficult to obtain in such a large-scale multicenter clinical study because of an uncompleted medical tracking system. In this study all 49 participating sites were referral centers mainly located in big cities, whereas pregnant women prefer to give birth in local hospitals, making follow-up assessment difficult. For these reasons, we were only able to obtain fetal outcome data on 4524 participants, even though we used all possible means to contact these women through both the participants centers and referring centers. Also, we came up with an incentive program to encourage these patients to report to us in case of false negative results. By introducing a formal insurance policy from a listed company, any reported false negative case would receive CNY 200,000 ($ ) of insurance coverage, which was equivalent to 8.3 years of the average household income in China (http://www.stats.gov.cn/). Therefore, it is unlikely that anyone with a false negative result would go unnoticed without making a claim to the insurance company. Because we have not received any claim for insurance so far, we believe that the chance of unreported false-negative case was very low. The sensitivity and specificity of over 99% for the MPS-based test for both trisomies 21 and 18 in this study was consistent with the results of previous studies. 21,22 Only 1.17% (190/11 105) of the study population which were classified as test positive for trisomy 21 or 18, needed to perform conventional karyotyping analysis, suggesting that over 98% of invasive procedures could be avoided. This is not only cost-saving, but also is a much safer option, avoiding unnecessary fetal losses because of invasive tests. If all of these pregnant women had received the invasive tests, 55 to 111 cases procedure-related miscarriage might have happened based on the 0.5% to 1% incidence found in prior studies. 3 5 These hypothetical miscarriages could be avoided with the introduction of our test to the established prenatal screening and diagnosis workflow. In principle, MPS-based methods could detect all kinds of fetal aneuploidy and microdeletions/microduplications. The testing for trisomy 21/18 is the most clinically relevant because other numerical autosomal chromosome aberrations are rare after 12 weeks On the other hand, the detection of other chromosomal abnormalities such as mosaic or structural variations still remains challenging for this new MPS-based test. Figure 4 Recommended pipeline to integrate the MPS-based test with established screening-diagnosis model in China. The pipeline with gray solid line refers to the established clinical procedures in China. The intensive route labeled with dark solid lines represents the preferred time of interference into conventional clinical practice. The dark dashed lines indicate our test can also be applied to pregnant women in the low-risk subgroup and those with negative results from conventional screening tests. Whether the MPS-based test is used as a primary or secondary screening test, it should be given careful pretest/posttest counseling based on informed consent and choice
7 Clinical application of sequencing-based prenatal noninvasive fetal trisomy test For the two false positive cases, one of the possible explanations is that there was microduplication in chromosome 21/18 that could be detected by karyotyping. Another possibility is that it was a placenta mosaic that was hard to detect by conventional invasive procedure. Further studies are needed to explore the application of the MPS-based test in these situations. With the revolutionary progress of noninvasive testing technology, new ethical questions are emerging. 32 One of the most difficult issues relates to the fact that such new approaches can count sex chromosomes and therefore contain information of fetal gender. At present, fetal sex is not reported in China because sex-selection is banned by the Law on Maternal and Infant Health of China. Would the availability of fetal sex information result in sex selection behavior? Because part of sex chromosomal abnormalities are not associated with major disabilities, should a woman be informed if there is a suspicion of sex chromosomal abnormalities? Thus, the key concern is what information could or should be provided to pregnant women. As for sex chromosomal abnormalities, it may be time to consider a more comprehensive informed consent process to allow pregnant women to make well-informed decisions on requesting this additional information or not. There are several limitations of the current study. First, the majority of subjects were recruited after 17 weeks of gestation, and only 371 pregnant women had a gestational age between 9 and 12 weeks, which may be related with the high accuracy of detection of trisomies 21 and 18 and low ratio of repeat test in this study. This was due to the fact that in most Obstetrics and Gynaecology departments in China, medical records for pregnant women begin from 12 gestational weeks, and this is also an important reason we introduce our test at this time point and beyond in this study. Further large scale studies focusing on subjects in the first trimester of pregnancy may be required to evaluate the performance of the MPS-based testing at an earlier gestation (9 12 weeks or even earlier) before its complete integration into current obstetrical practice. Second, although the inadequacy of clinical follow-up could partially be overcome by the use of an insurance coverage scheme, further long-term investigations still need to be carried out for more comprehensive evaluation of this new technology. CONCLUSION Massively parallel sequencing-based test as a screening method is undoubtedly more efficient than any other existing noninvasive screening tests for fetal trisomies 21 and 18. Although wide-spread use of this technology is limited by the cost and reporting time at the moment, the situation probably will change quickly. The cost of MPS-based noninvasive test varies between countries, even states. In the United States, this MPS-based test is charged $795 to $2762, while it takes around $500 to $1000 in China. With the rapid development of high throughput sequencing technology, the cost of this test will drop to levels lower than that of conventional invasive diagnosis procedures in the near future. The reporting time of the MPSbased test could be reduced to three days or less. 33,34 Also, with increasing sequencing depth, our test has the potential to detect more fetal chromosomal abnormalities including structural variations or even single-gene disorders It is clear that this new technology will radically change the current clinical model of prenatal screening and diagnosis in the near future. However, the proper application of the MPS-based test in clinical settings needs careful consideration to integrate with established obstetric practice and workflow, and the right of informed consent and choice of pregnant women should be fully respected in this process. Here we also recommend a possible integration of the MPS-based test with the established screening-diagnosis model based on our experience in China (Figure 4). WHAT S ALREADY KNOWN ABOUT THIS TOPIC? Massively parallel sequencing has been proved to be feasible for the noninvasive prenatal diagnosis of trisomy 21. This is a large scale multicenter study of massively parallel sequencing-based noninvasive prenatal testing for trisomies 21 and 18 in China. WHAT DOES THIS STUDY ADD? We proved that our noninvasive fetal trisomy test is successful as a screening test for detection of trisomy 21 and 18 in the Chinese population. REFERENCES 1. Statistics NBo. The population change of China in 2011 [WWW document]. 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Support and Information Rare Chromosome Disorder Support Group, G1, The Stables, Station Rd West, Oxted, Surrey. RH8 9EE Tel: +44(0)1883 723356 firstname.lastname@example.com I www.rarechromo.org Balanced UniqueMore information
CONGENITAL HEART DISEASE Introduction Congenital heart disease (CHD) is the most common congenital disorder in newborns [1]. Due to definitional issues, there are large variations in prevalence estimates.More information


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Our Team
We invite you to explore the biographies of our VCA Southern Maryland Veterinary Referral and Emergency Center's veterinarians and staff and learn more about them. Our hospital's team of doctors and staff are compassionate and highly trained veterinary professionals and specialists who are ready to assist you and your pet whenever a health concern presents itself. As you will see in our biography section, our staff has a wide variety of backgrounds and interests, but, above all, a love of animals and a commitment to providing the very best care for them.
The Staff of VCA Southern Maryland Veterinary Referral and Emergency Center.

subset: commoncrawl


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Soon you will be able to unleash the mark of Zorro with your Wii Remote. The classic hero makes his video game debut this spring in The Destiny of Zorro from 505 Games.
Players will make swashbuckling use of the motion-sensitive remote in the role of Zorro, whose real identity is Don Diego de la Vega, a defender of the lowly in early 19th century Spanish California. The new storyline has Zorro doing battle against nemesis Calavera, a mob boss and an entire rebel army in the various missions.
"We are excited to extend the global phenomenon of Zorro from movies, live action and animated television series, and our Olivier Award-winning musical, to the video game world," said Zorro Productions CEO John Gertz in the announcement. "With videogame technology, we're able to put fans and gamers in the middle of the action, to experience the fantasy of Zorro in a whole new way."
By Mike Snider
Image courtesy of 505 Games
Mike Snider began covering the video game industry during the Super Nintendo-Sega Genesis clash in 1992. An original pinball wizard, he eventually was seduced by Robotron: 2084 and Tempest. These days he is a fan of action/shooters and lives out his Keith Moon fantasies playing a mean drum kit on music games. More about Mike.
Brett Molina has been writing about video games for USA TODAY since 2005. He is well-versed in Madden NFL, the fighting genre and first-person shooters. The first video game he played was Asteroids at a local arcade. He has been hooked ever since. More about Brett.


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Am I too thin for a BBL? (Photo)
Doctor Answers 8
BBL candidate
Too Thin for BBL?
Learn more: http://www.bodysculpt.com/brazilian-butt-lift.html
You might also like...
Am I too thin for a BBL? (Photo)
Learn more: http://spectrum-aesthetics.com/brazilian-butt-lift/
Am I too thin?
Not Too Thin
In my practice we usually take from the back to sculpt out and give that perfect A shape vs the V shape some women have. We also take from additional areas if necessary or requested by the patient. You already have a great A shape, but it can be accentuated. Based on the amount of fat your surgeon of choice is able to harvest will determine how large you are able to go. From your photos provided there are definitely some donor sites that your surgeon would be able to pull the adequate amount of injectable fat from.
One rule of thumb I like to go by is, if you start with a great shape you will end up withe great results! I wish you the best of luck on this exciting journey!
**"The above answer posted has been given without seeing this patient and cannot be substituted for an actual in-person examination/consultation with a medical professional and professional advice; it is only opinion based on photos and/or description of symptoms. Seek in-person treatment with a trained BCPS medical professional for appropriate action and care to be given."**
Learn more: http://nuvistaplasticsurgery.com/
Brazilian Butt Lift/ Fat grafting/Liposuction/ High Definition Procedures/Butt Implants
Learn more: http://beverlyhillsbreastandbodyinstitute.com
Am I too thin for a BBL?
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


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The new look site offers stunning visuals to propel you into the virtual gaming arena of a world class Casino lobby.
The online poker room still offers all the same exciting play options as before with lots of new features added including a more user-friendly site structure.
As part of the revamp there is a new loyalty scheme which means that the more you play, the more you can earn to play at the tables.
Player statistics will allow players to keep track of their bankroll and also to see how much rakeback they've accumulated.
There is now also access available to the exclusive backstage area, which offers the kind of glamour and VIP treatment one would experience at a top class Casino.


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How much do MIT employees make in Cambridge, MD? Glassdoor has salaries, wages, tips, bonuses, and hourly pay submitted anonymously by 119 MIT employees in Cambridge, MD.
"It's an HMO plan. No matter what they try to tell you, their most expensive plan is still an HMO (not PPO) plan. MIT Medical? Forget it. Most..."
"Very good on the whole, but at beginning of fall and spring semester, can be hard to get. Be sure to ask wellin advance."
MIT Salaries by Location
Check out the latest MIT Jobs & Careers


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Watching a movie on the big screen makes audience members feel like they're really there with the characters, especially during climactic popcorn-eating scenes. Chew and view with this Groupon.
Choose Between Two Options
- $10 for a movie package for two (up to a $23 value)
- $19 for a movie package for four (up to a $46 value)

Each movie package includes the following:
- One movie admission per person (up to a $3.50 value each)
- One medium popcorn per person (a $4 value each)
- One medium soda per person (a $4 value each)

Moviegoers can head to the Denver, Arvada, or Littleton theater to catch second-run family-friendly flicks such as Ice Age: Continental Drift and Brave, or the latest installments in the Bourne and Batman movies, The Bourne Legacy and The Dark Knight Rises. Alternatively, they can check the coming attractions and wait for the arrival of flicks such as the animated Wreck-It Ralph.

Elvis Cinemas' ticket prices fluctuate throughout the day.
Elvis Cinemas
Elvis Cinemas invites wide-eyed audiences to plunge into the ocean, soar above the clouds, and traipse across stretches of barren desert, all from the comfort of a theater seat. Its trio of theaters shows Hollywood blockbusters but keeps ticket prices down by playing them slightly after their initial release, when the characters have learned from their mistakes and changed their movie's plot accordingly. Unlike the massive movie corporations whose theater complexes pop up in every city and shopping mall, Elvis Cinemas is Colorado owned and operated and focuses all its attention on just three theaters.


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Edit Page Last Edit:
EditUltimate MvC3 Changes
EditOfficial Changelog
- Mid-air special attacks can be canceled into Talon Attack.
- Crescent Scythe floats higher.
- Decreased untechable time for opponents after a Crescent Scythe hit.
- Rage Trigger causes opponent to fly further to the side.
- Number of hits in Rage Trigger can be increased through rapid buttons presses.
- Rage Trigger hyper starts up more quickly, makes for easier OTG combos
- Crescent Scythe can cancel into Talon Attacks
EditHow to Play
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Continuous improvement, based on Japanese concept called KAIZEN, is the philosophy of continually seeking ways to improve operations. It involves identifying benchmarks of excellent practice and instilling a sense of employee ownership of the process. (more…)
September 1, 2009
March 18, 2009
Kaizen: The Life Blood of Standardized Work
Standardized work and KAIZEN are the means by which people make the Toyota Production System work. People are by far the most important element of the entire system. Without the support of everyone involved, no part of the system will work. No matter how ingenious the method of production or service may be, for example, if the workers do not follow rules, the entire system of production control will fall apart. (more…)


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2nd Annual Champagne & Bubbles Extravaganza!
Wine World & Spirits
Saturday, December 7, 2013 from 2:00 PM to 5:00 PM (PST)
Who's Going
San Francisco, California
London, United Kingdom
Florence, Italy
Event Details
Come celebrate Wine World & Spirits' Three Year Anniversary in style with a Champagne & Bubbles Extravaganza!
From 2 to 5 pm, there will be champagne and bubbles tastings in the Wine World Ballroom!
We will have over 14 stations pouring Champagne and sparkling wine!
Come enjoy champagne, bubbles, and light nibbles as we celebrate Wine World's success due to our fantastic community partners and your loyal support of a local business. Please Share this event and tell your friends to come!!!
When & Where
Wine World & Spirits
400 NE 45th St
Saturday, December 7, 2013 from 2:00 PM to 5:00 PM (PST)
Add to my calendar
Wine World & Spirits
Largest wine shop in the Northwest with a great selection of beers from near and far.


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Insurance organizations
It should be noted the fact that the number of Bank and non-Bank financial institutions ready to provide its clients with cash now number in the thousands and all such organizations, according to advertising slogans and avenues, only exclusive, profitable and reliable credit conditions. In other words, to pay the loan all heirs have accepted the inheritance, to avoid this will not succeed.
The insurance entities
Moreover, the loyalty of financial institutions in creating these tandem can reach such proportions that they are ready to offer our potential borrowers loans at favorable interest rate, which will be issued against collateral in the form of real estate under construction. Very often potential borrowers are lured by the fact that promise to provide consumer loans or cash loans within 15 minutes at the time of treatment, and from the potential borrower in obtaining such loans will not need to provide any documents other than passports.
The insurance entities - actuaries, insurance brokers, mutual insurance societies and insurers. Insurance organizations - insurance companies life insurance, insurance companies for insurance other than life insurance and reinsurance companies.
mutual insurance
mediation activity


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Thank you for taking the time to explore Paragon Homes. For over 20 years, we have offered clients outstanding service and exceptional value. Our homes have spanned from villa-styled single-family to spacious luxury Estates. Our goal is to understand our clients needs and exceed their expectations. The result is an affordable, simple and enjoyable building experience.
- Category: Business Cards
- Tags: burgh custom home builder


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Liona Bee – Liona likes Leather
Released: December 26, 2016
Best Liona Bee Porn Videos (7)You just found 7 Liona Bee porn videos collection ! Naughty Blog has the hottest Liona Bee videos for Free and you can Download them right now. Our users love this pornstar. Check out our 7 sexy Liona Bee porn videos below. Like what you see? Make sure to share our page and sign up for RSS Feed ! Share RSS
Teen Bitch Club – Liona Bee
Dec 26
Liona Bee – Liona likes Leather
Liona Bee – Shooting the Perfect Load
Released: November 2, 2016
Liona Bee has the perfect body, and as our photographer tries to capture her beauty, he feels the need for something more than a still image. As she undresses, he cannot resist, and must taste this perfection from head to toe. They embrace, and he soon penetrates his favorite model in ways never before felt. The anal ecstasy culminates in a perfect climax, and as she moans with delight he finishes all over her immaculate bottom.
The Art Porn – Liona Bee
Oct 30
Liona Bee – High on your love
Released: October 30, 2015
The couple in this beautiful and unique erotica video doesn't like going out. They don't really enjoy clubs, restaurants and dinners with friends. All they ever want is to be home, just the two of them. Some might think it's so boring, but wait until you watch this steamy glam porn video till the end! These two hardly ever stop making love! They feel horny all the time, and they can't get enough of each other. Here's your chance to admire the neverending lust of our zealous lovebirds and to take pleasure in their heated lovemaking.
Anal Teen Club – Liona Bee
Mar 27
Liona Bee – Casting Couch
Released: March 27, 2015
Liona Bee is a sweet little teen who wants to prove herself in front of the cameras. But to achieve that and get her own reputation, she has to demonstrate her skills first. Today we gave her the chance to show what hides under that sexy attire and more importantly, what kinky thoughts are behind that silky forehead.
Liona Bee – Anal Teen Angel Liona Bee
Released: March 2, 2015
Anal-obsessed Liona Bee wanted to come back for another turn of ass-banging fun. She was so eager and sweet (and sexy too) that we couldn't say no. How could we to one of our loveliest Anal Teen? So Liona is back to have some fun and to entertain you with some hot anal sex.
Irina Pavlova & Liona Bee – Sexual Curiousity
Released: December 25, 2014
Irina and Liona are two cute kittens who like to play with each other to explore their own bodies, their own lust, to fulfill their own fantasies. And what could be the safest way to do it than practicing on each other? Curious lips, tongues and fingers wander around of tight, silky young bodies for everyone's greatest satisfaction.
Anal Teen Angels – Liona Bee
Dec 15
Released: December 15, 2014
Let us introduce our newest Anal Teen Angel. Her name is Liona Bee and she is a really cute and eager minx with a lot of joy and horny desire in her slender, lovely frame. She also LOVES anal sex, with all capitals, which makes her an excellent girl to be one of the Angels. Welcome her with as much love as we did.


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By Mike Dodd, USA TODAY
CHICAGO Wearing their 1940s Monsters of the Midway uniforms, the Chicago Bears flashed their old playoff form Monday, defeating the Green Bay Packers 20-17 to improve their record to 3-0 for the first time since the Super Bowl season of 2006.
Linebacker Brian Urlacher forced a pivotal fumble late in the fourth quarter and Devin Hester returned his first kick for a touchdown in more than two years as the Bears became the only remaining unbeaten team in the NFC.
The Packers (2-1) helped Chicago's cause greatly with a team-record 18 penalties for 152 yards, including a holding call that negated a Green Bay touchdown and two defensive personal fouls on the Bears' game-tying field-goal drive late in the fourth quarter.
RAY LEWIS: Packers should have let Bears score
Urlacher's strip of receiver James Jones and cornerback Tim Jennings' recovery of the fumble with 2:18 remaining gave the Bears the ball on the Green Bay 46, setting up Robbie Gould's 19-yard field goal with four seconds left for the victory.
Earlier in the quarter, Hester, the record-breaking return man turned wide receiver, broke a two-year TD return drought when he scooted 62 yards for a touchdown on a punt return to give the Bears their first lead of the game. It was his first return for a TD since Dec. 30, 2007.
BOX SCORE: Bears 20, Packers 17
BIG RETURN: Hester's 12th return TD is one off record
PHOTO GALLERY: Highlights from Week 3 in the NFL
"When we honor the Monsters of the Midway, it was just good to see a team just play 60 minutes of ball," Bears coach Lovie Smith said. "The guys just weren't going to be denied."
Chicago's Jay Cutler, sacked three times and pressured heavily at others, completed 16 of 27 passes for 221 yards with one touchdown and one interception. Another interception was called back by a roughing the passer penalty.
"We didn't play our best game and we won," Cutler said. "I felt we were out of sync. There were some throws I should have made. The defense did a great job, we struggled the whole game, felt like we kind of were killing ourselves."
Green Bay quarterback Aaron Rodgers, without the benefit of much of a running game, completed 34 of 45 passes for 316 yards, one TD and one interception. His 3-yard touchdown scramble with 6:52 left gave the Packers a 17-14 lead.
But the Bears, with the benefit of two defensive personal fouls, went 67 yards to set up Gould's tying 25-yard field goal with 3:59 left.
Green Bay started marching down the field again, and Rodgers hit Jones for a 12-yard gain to midfield. Linebackers Lance Briggs and Urlacher converged on the receiver and Urlacher, playing to his All-Pro form of a few years ago, reached in and stripped the ball for the game-changing turnover.
"We're 3-0 because our top players, our marquee players, are playing that way," Smith said.
The Packers led throughout the first three quarters after jumping to a 7-0 lead on their first possession when Rodgers hit Greg Jennings with a 7-yard TD pass.
The wild fourth quarter started when Hester, who nearly broke a return for a TD in the second quarter only to be stopped by punter Tim Masthay, took a punt at the Chicago 38 on the second play of the quarter, split two Green Bay defenders and took off. He leaped over a diving Masthay at the Packers' 5, determined the punter wasn't going to bring him down a second time.
"It's been a long time," said Hester. "I hope I proved that I'm still capable of doing it."
Green Bay coach Mike McCarthy lamented the team's penalties. "Huge factor in the outcome of the game," he said. "It's hard to play football like that."
Former all-pro defensive tackle Tommie Harris was inactive for the game and the Bears said it was a coach's decision. "Performance-based," Smith said.
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text: It was not by SturgeonsLaw that these fanfics are once again given mention. They were added here by ''humans'' who wish to pay them tribute.These are recommendations made by Tropers for ''Franchise/{{Castlevania}}'' {{fanfic}}s, all of which have to be signed to stay on the page. Feel free to add a fanfic of your own to the list, but remember to use the template found [[FanficRecommendations here]].Do warn when a fanfic may head into sexual or [[BetterThanCanon non-canon]] territory. Some people just don't like it, and as we all know, {{Shipping}} is SeriousBusiness. You can also add to the current recommendations if you want. Refrain from posting Administrivia/ConversationInTheMainPage though; that goes in the discussion page.----[[WMG: Authors, and Websites]]None yet.[[WMG:[[GenFic General Fics]]]]''Stories focused on the family and the friendly relationships of the cast. Plot-focused stories or light day-in-the-life stories. Pretty much anything that isn't focused on romance.''''[[http://www.fanfiction.net/s/5483316/1/Castlevania_All_His_Engines Castlevania: All His Engines]]'' by Legend Maker* Recommended by Gamerex27, seconded by Tropers/{{Yuihime}}, thirded by Kazeto* Synopsis: "For centuries, Dracula has warred upon humanity. For centuries, Dracula has been certain in their inferiority. For centuries, Dracula has never been proven wrong. Dracula is about to get a different answer to his question of what a man is..."* Comments: This is an amazing oneshot, with some of the most incredible characterization that this troper has ever seen. It really delves into the mind of Dracula, and the [=OCs=] are all believable and realistic by Castlevania's standards.** Kazeto: While the story is not what I had been hoping for when I had stumbled upon this fiction (and at that time I was looking for heavily psychological and/or philosophical fictions, with this fiction not being heavy), it is still a very pleasant read. I recommend wholeheartedly to every person familiar with Castlevania lore.''[[http://www.fanfiction.net/s/7153898/1/Castlevania-Nocturne-of-Ruin Nocturne of Ruin]]'' by Draconic* Recommended by Tailikku* Synopsis: "Dracula's castle has risen again, and dark forces are at work. It's up to Jonathan, Charlotte, the Lecarde sisters, and two familiar friends to traverse Castlevania again and put an end to the horrific plans of an almost as familiar enemy from ages past."* Comments: Excellently written. This story manages to act as a sequel to [[VideoGame/CastlevaniaPortraitOfRuin Portrait of Ruin]] by including elements from other games, notably VideoGame/CastlevaniaHarmonyOfDespair, [[VideoGame/CastlevaniaSymphonyOfTheNight Symphony of the Night]], and [[VideoGame/CastlevaniaLamentOfInnocence Lament of Innocence]]. Also pokes fun at bad translations from previous games and makes nods towards the horror films that inspired the series in the first place.* Status: Complete* [[http://tvtropes.org/pmwiki/pmwiki.php/FanFic/CastlevaniaNocturneOfRuin Has its own tropes page]] currently in progress.''[[https://www.fanfiction.net/s/4754974/1/Forgotten Forgotten]]'' by Hotaru-hime* Recommended by Draconic* Synopsis: "Mina POV. For love, he declared war against God. For love, he declared war against mankind. What would he do if he ever lost her again?"* Comments: A beautiful story that elegantly ties [[VideoGame/CastlevaniaAriaofSorrow Aria of Sorrow]] and [[VideoGame/CastlevaniaDawnofSorrow Dawn of Sorrow]] directly to [[VideoGame/CastlevaniaLamentOfInnocence Lament of Innocence]] and onward to [[VideoGame/CastlevaniaSymphonyOfTheNight Symphony of the Night]]. It acts as a sequel to [[VideoGame/CastlevaniaDawnofSorrow Dawn of Sorrow]] as well, making it perfect for fans of Soma, and as it's told from Mina's perspective, her fans will fall in love with this fic very quickly. It also features Julius, Yoko, Arikado/Alucard, and even Hammer; the entire cast of protagonists from Aria & Dawn, really. It displays Soma's connection to Dracula through Mina's point of view in a ''very'' clever way, as well as drawing a very sympathetic Mathias/Dracula, even while showing his ruthlessness as a vampire.* Status: Complete''[[https://www.fanfiction.net/s/10489396/1/Rebirth Rebirth]]'' by Orionali* Recommended by: eagle108* ''Status:'' Complete* ''Synopsis:'' Zobek is dead, Lucifer has been vanquished, Vampire Killer recovered, Wygol saved. What awaits the Prince of Darkness and his Heir now? A Reverie, a Resurrection, a Revelation, a Rebirth.* ''Comments:'' The blending of additional biblical themes, as well expanding on [=LoS=]'s background lore paints a stronger realm in ''Rebirth'' here as Dracula goes down an unfamiliar path with Alucard, some of which could fall under FridgeBrilliance when taken in context.''[[https://www.fanfiction.net/s/10504861/1/Blood-Is Blood Is]]'' by eiahmon* Recommended by: eagle108* ''Status:'' Complete* ''Synopsis:'' [[AlternateUniverse AU]]. Trevor wasn't the only one who had the truth of his parentage kept from him by the Brotherhood.* ''Comments:'' What happens if the rumors that Gabriel Belmont was the son of the noble Conrqvist family were actually true? This fic answers that question and how the events after the first game could have diverged greatly.''[[https://www.fanfiction.net/s/11368601/1/One-Light-Burning One Light Burning]]'' by eiahmon* Recommended by: eagle108* ''Status:'' Complete* ''Synopsis:'' [[AlternateUniverse AU]]. When Alucard wakes in his sarcophagus, he doesn't look at the mirror and so does not regain his memory of what had happened to him. Instead he finds his father, and the entire world shifts.* ''Comments:'' It's an interesting take on "what if" Alucard never knew what he was [[spoiler: courtesy of the Mirror of Fate/the Lost Soul showing what had happened]], and the few actual interactions he has with Simon Belmont.''[[https://www.fanfiction.net/s/10126863/1/Red-Road Red Road]]'' by Cosmic Sky* Recommended by: eagle108* ''Status:'' Complete* ''Synopsis:'' [[AlternateUniverse AU]]. Before Simon handed Alucard the mirror shard he looked into it and saw what happened to Gabriel and Trevor. His thoughts and questions on his family legacy and what he intends to do carrying on Belmont the name.* ''Comments:'' It originally started off as a one-shot of Simon, but each chapter is a reflection of various characters through the trilogy.[[WMG:[[{{Shipping}} Shipping Fics]]]]''Stories focused on the romantic relationships between the cast.''None yet.[[WMG: [[{{Crossover}} Crossover Fics]]]]''Stories that blend Castlevania with another series.''''[[http://www.fimfiction.net/story/46026/equestrylvania Equestrylvania]]'' (formally "My Little Castlevania") by Brony_Fife* Crosses Over With: WesternAnimation/MyLittlePonyFriendshipIsMagic* Recommended by bifferboffer* Synopsis: Canterlot Castle has vanished overnight, and a strange new castle has taken its place. Upon investigating, Twilight Sparkle finds that a new evil has been unleashed upon Equestria. Does she and her companions stand a chance against the evil of Count Dracula?* Comments: Instead of going for action, the author instead heads into a straight-up horror story. While there is plenty of action, there is much more in the way of scares.* [[Fanfic/{{Equestrylvania}} Now has it's own page]].''[[http://www.fanfiction.net/s/8479671/1/Lyrics-of-Sorrow Lyrics of Sorrow]]'' by [=LoneWolf218=] ([[FanFic/LyricsOfSorrow TV Tropes page]])* Crosses Over With: ''Anime/MagicalGirlLyricalNanoha''* Recommended by: {{Tropers/SAMAS}} and Draconic* Synopsis: Soma Cruz is called to investigate the cause of a sudden unnatural eruption of plant life. He soon finds himself in the middle of a struggle between children for artifacts of unimaginable power, artifacts more local forces of darkness start to take an interest in. ''Fanfic/QueenOfBlood'' by SirWill* Recommended by nitewind, WaltzBurgNacht* ''Crossover with:'' {{Literature/Worm}}* ''Status:'' In progress* ''Synopsis:'' Taylor Hebert dies in the locker. Then she wakes up in her grave, changed. Powerful. With faint memories of a life not her own. And why does she dream of a castle in the mountains?* Fanfiction.net link: https://www.fanfiction.net/s/11698925/1/Queen-of-Blood''[[https://www.fanfiction.net/s/10832798/1/Anthology Anthology]]'' by Iniora Nackatori* Recommended by: eagle108* Crosses over with: ''Series/BuffyTheVampireSlayer''* ''Status:'' Complete* ''Synopsis:'' Buffy Summers, daughter of Joyce Summers, granddaughter of... Vincent Belmont. Fate didn't pick Buffy to be the Slayer just because; it picked her to screw with her bloodline. Which is something her great-to-the-n'th-degree grandpa Gabriel is Not Amused with...----


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This property is no longer available.Search for Available Property.
1 acres in Jackson County, Florida
Wooded Residential Lot of over 1 Acre in the Compass Lake in the Hills development in NorthWest Florida. Conveniently located just 20 minutes South of Interstate 10 at Marianna, and 40 minutes North of Panama City for Shopping, Restaurants, Entertainment, and Beautiful Gulf Beaches. As an owner in this development you have access to Private McCormick Lake for Fishing, Swimming, Canoeing, Kayaking, etc as well as the Main Lodge over on Compass Lake with Swimming Pool, Tennis Courts, Horseback Riding, etc. Come enjoy the Quiet, Peaceful Lifestyle of Compass Lake in the Hills.
From the intersection of Hwy 231 and Hwy 20 travel North on Hwy 231 for 7.3 mi then Right onto Co Rd 167 for 6.5 mi then Right onto Woodla for 1/4 mi ( jog Left then Right ) then Right onto Jackson for 1/4 mi then Left onto Cove and Lot #4 will be 500 feet on your Right ( South side of Cove Ave
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NOTE: The information contained herein is provided as general information only and has been obtained from sources deemed reliable. It is provided without any guaranty, warranty or representation, expressed or implied, made by Lands of America, or any related entity, as to the accuracy or completeness of the information. The information is presented subject to errors, omissions, change of price or conditions, prior sale or withdrawal without notice. Prospective purchasers should make their own investigations, projections and conclusions concerning the information.


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Jadi saya datang nak sapu habuk dan sarang labah2 yg dah banyak kat sini.
so, just a few updates from me.
1. i am now at shah alam doing master in accountancy part time. so the class will be on saturday and sunday only.
2. i am working at shah alam as account and admin assistant.
3. i am still single and waiting for my mr. right.
ok. That's all for now. see u guys later.


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House/TechHouse Dj-Producer From Argentina.
Contact - Info - Booking: firstname.lastname@example.com
Huambo Records
Clover Records
Bedroom Muzik
Soulfreak Records
Rhombus Digital Records
Vayk Records
Wanna Dance Music
Flagman Records
And many others...
Listen to all music and sounds of Joph Wa and follow this user by signing in for free.
You can listen to 5 tracks and mixes and follow this user as 4 did before.
This artist liked 8 tracks, followed 4 others and reshared 0 tracks and mixes. 5 other people are following this page too.
soundcloud...om/joph-wa mixcloud.com/JophWa instagram.com/jophwa


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Oh most powerful St Jude, you have helped me in the past and here I am seeking your miraculous intercession one again. Please give my sons the courage and strength to fight their addictions, and help them to become honest and trustworthy men.
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