Cytogenetics Laboratory Research Articles

Chromosomal abnormalities and polymorphisms among couples with recurrentin vitrofertilization (IVF) failure

Aim: The present study aims to evaluate chromosomal structure among couples with a history of recurrent in vitro fertilization (IVF) failure undergoing assisted reproduction treatment. Materials and Methods: Chromosomal structure analysis was conducted on peripheral blood samples from couples with an indication of recurrent IVF failure at the Genetic Diagnosis Center Cytogenetics Laboratory between 2007 and 2012. All data were investigated retrospectively. Results: Abnormal chromosome organization was observed in 39 (15.8%) females and in four (1.7%) males. Moreover, chromosomal polymorphism was detected in 15 (6%) females and 28 (12%) males. Almost all chromosomal abnormalities were associated with sex chromosomes. Conclusion: Highly abnormal chromosome organization was observed in couples with recurrent IVF failures. Preimplantation genetic diagnosis should be offered to these patients to evaluate the likelihood of treatment success and the establishment of a healthy pregnancy.

Changes in and Efficacies of Indications for Invasive Prenatal Diagnosis of Cytogenomic Abnormalities: 13 Years of Experience in a Single Center.

BackgroundBecause the future application of cell-free fetal DNA screening is expected to dramatically improve the diagnostic yield and reduce unnecessary invasive procedures, it is time to summarize the indications of invasive prenatal diagnosis. This retrospective study was performed to evaluate the changes and efficacies of indications of invasive procedures for detecting cytogenomic abnormalities from 2000 to 2012.Material/MethodsFrom our regional obstetric unit, 7818 invasive procedures were referred by indications of advance maternal age (AMA), abnormal ultrasound findings (aUS), abnormal maternal serum screening (aMSS), and family history (FH). Chromosome, fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (aCGH) analyses were performed on chorionic villus sampling (CVS) and amniotic fluid (AF) specimens at the Yale Cytogenetics Laboratory. The abnormal findings from single or combined indications were compared to evaluate the diagnostic yield.ResultsThe annual caseload declined by 57.2% but the diagnostic yield increased from 7.2% to 13.4%. Chromosomal and genomic abnormalities were detected in 752 cases (9.6%, 752/7818) and 12 cases (4%, 12/303), respectively. Significantly decreased AMA referrals and increased aUS and aMSS referrals were noted. The top 3 indications by diagnostic yield were AMA/aUS (51.4% for CVS, 24.2% for AF), aUS (34.7% for CVS, 14.5% for AF), and AMA/aMSS (17.8% for CVS, 9.9% for AF).ConclusionsOver a period of 13 years, the indication of aMSS and aUS were increasing while AMA was decreasing for prenatal diagnosis of cytogenomic abnormalities, and there was a continuous trend of reduced invasive procedures. Prenatal evaluation using AMA/aUS was the most effective in detecting chromosomal abnormalities, but better indications for genomic abnormalities are needed.

A high-resolution copy-number variation resource for clinical and population genetics.

Chromosomal microarray analysis to assess copy-number variation has become a first-tier genetic diagnostic test for individuals with unexplained neurodevelopmental disorders or multiple congenital anomalies. More than 100 cytogenetic laboratories worldwide use the new ultra-high resolution Affymetrix CytoScan-HD array to genotype hundreds of thousands of samples per year. Our aim was to develop a copy-number variation resource from a new population sample that would enable more accurate interpretation of clinical genetics data on this microarray platform and others. Genotyping of 1,000 adult volunteers who are broadly representative of the Ontario population (as obtained from the Ontario Population Genomics Platform) was performed with the CytoScan-HD microarray system, which has 2.7 million probes. Four independent algorithms were applied to detect copy-number variations. Reproducibility and validation metrics were quantified using sample replicates and quantitative-polymerase chain reaction, respectively. DNA from 873 individuals passed quality control and we identified 71,178 copy-number variations (81 copy-number variations/individual); 9.8% (6,984) of these copy-number variations were previously unreported. After applying three layers of filtering criteria, from our highest confidence copy-number variation data set we obtained >95% reproducibility and >90% validation rates (73% of these copy-number variations overlapped at least one gene). The genotype data and annotated copy-number variations for this largely Caucasian population will represent a valuable public resource enabling clinical genetics research and diagnostics.

Population Based Study of Cytogenetic Abnormalities in Addition to Philadelphia (Ph) Chromosome in Patients with Chronic Myeloid Leukaemia (CML) and Impact on Survival

Ph chromosome is the hallmark of CML. However there are few reports of additional chromosomal abnormalities at the time of diagnosis and the impact this has on overall survival (OS). [NT1] The Cytogenetic laboratory at this hospital provides a regional service as a single facility. The data from this laboratory was combined with survival information to evaluate the impact of additional chromosomal changes on outcomes in patients with CML.Methods: This is a retrospective population based study, it was not possible to obtain consent from individual patients and details about haematological parameters or treatments delivered were not available. The aim was to evaluate if cytogenetic changes should be considered in addition to established risk scoring systems. Patients were classified as complex-Philadelphia (Ph) if they had t(9;22)(q31;q34) with additional chromosomal abnormalities. Impact of individual additional abnormalities was analysed and then the effect was stratified according to presence of chromosomal gains, deletions or translocations. Few cases who had normal cytogenetics on their first sample as they were initially treated elsewhere.Results: 1129 patients were diagnosed with CML between 1985 and 2013, with 4760 samples analysed. The median age of the patient was 52.4 years (4.3-103, 602 male; 511 female; 16 unknown). Median follow up was 6.4 years [0-26.8 years, 725/1129 (64.2%) had follow-up more than 10 years]. End point for analysis was probability of survival at 10yr. 194/1129 (17.2%) had complex-Ph at diagnosis, 759/1129 (67.2%) had standard Ph, 77/1129 (6.8%) had negative cytogenetics and in 34/1129 (3%) cytogenetic analysis failed at diagnosis. Patients with standard Ph translocation had significantly better chance of achieving cytogenetic CR than those with complex-Ph (23.4% vs. 13.4%, p<0.001). OS was significantly better in patients below the age of 45 (65% vs 25% p <0.0001). OS was also better in patients diagnosed after 2000 (67 % vs 40 %, p<0.0001). In univariate analysis OS was significantly lower with trisomy 8 (10% vs 50%, p<0.0001), del(5q) [NT2] (20% vs 50%, p=0.001), other deletions (12% vs 48%, p=0.0005), del 17( 48% vs. 0%, p<0.0001), add 21 (50% vs. 0%, p<0.0001), any translocations (50% vs. 22%, p<0.0001), der 22 or iso 17 (48% vs. 10%, p<0.0001), any deletions (50% vs. 20%, p<0.0001) and variant Ph translocations (50% vs 22%, p=0.004). In multivariate analysis, excluding year of diagnosis, age group (HR 1.93, 95% CI:1.6-2.4 P=<0.0001), complex t(9;22;v) (HR 1.8, 95% CI:1.0-3.1, P=0.035), del(17q) (HR 3.8, 95% CI:1.1-12.6, P=0.033), translocations (HR:1.6, 95% CI: 1.1-2.25, p=0.013), trisomy 8 (HR: 1.76, 95% CI: 1.19-2.65, p=0.005), add 21 (HR: 3.21, 95% CI: 1.65-6.25, p=0.001) and der 22 or iso17 (HR: 1.57, 95% CI: 1.1-2.25, p=0.013) were independently associated with inferior OS. Number of risk factors in individual patients was used to design a scoring system. Patients with 0 risk factor (70% OS at 10 yr.), 1 risk factor (40% OS at 10 years), 2 risk factors (22% OS at 10 years) or more than 3 risk factors (12% OS at 10 years) had incrementally reduced OS. This was true of patients diagnosed before and after 2000.This analysis suggests that incorporating nature of karyotype at diagnosis can refine established scoring systems. However this data needs to be analysed with larger patient population to include all established risk factors and the effect of therapeutic measures. DisclosuresCavet:Novartis: Research Funding; BMS: Research Funding.

Reagent and Labor Cost Optimization through Automation of Fluorescence In Situ Hybridization (FISH) with the VP 2000: An Italian Case Study

In the modern molecular diagnostic laboratory, cost considerations are of paramount importance. Automation of complex molecular assays not only allows a laboratory to accommodate higher test volumes and throughput but also has a considerable impact on the cost of testing from the perspective of reagent costs, as well as hands-on time for skilled laboratory personnel. The following study tracked the cost of labor (hands-on time) and reagents for fluorescence in situ hybridization (FISH) testing in a routine, high-volume pathology and cytogenetics laboratory in Treviso, Italy, over a 2-y period (2011-2013). The laboratory automated FISH testing with the VP 2000 Processor, a deparaffinization, pretreatment, and special staining instrument produced by Abbott Molecular, and compared hands-on time and reagent costs to manual FISH testing. The results indicated significant cost and time saving when automating FISH with VP 2000 when more than six FISH tests were run per week. At 12 FISH assays per week, an approximate total cost reduction of 55% was observed. When running 46 FISH specimens per week, the cost saving increased to 89% versus manual testing. The results demonstrate that the VP 2000 processor can significantly reduce the cost of FISH testing in diagnostic laboratories.

Cytogenetic and clinic evaluation of two cases that have 45,X/46,X,i(Xq) and 46,X,i(Xq) karyotype

In this study, cytogenetic and clinic findings of 46,X,i(Xq) and 45,X/46,X,i(Xq) which are the rare types of Turner syndrome are evaluated. Two patients were directed to the Cytogenetic Laboratory from Gynecology Clinic with chromosomal anomaly indication. Their peripheric blood samples are analyzed karyotypically using Giemsa-Tripsin Bantama method. Case 1. The patient is 17 years old, has short height, a low-posterior hairline, short neck, small and wide apart breasts, normal external genitals and underdeveloped internal genitals. Her karyotype was 45,X/46,X,i(Xq). Case 2. The patient is 19 years old, has primer amenore, short height, a low-posterior hairline, wide apart and underdeveloped breasts, normal external genitals, underdeveloped uterus, obscure ovarium. Her karyotype was determined to be 46,X,i(Xq). Although symptoms progress slightly weaker, our 45,X/46,X,i(Xq) and 46,X,i(Xq) cases, which are rare types of Turner Syndrome, are generally consistent with phenotypic findings of Turner syndrome.

Pasado, presente y futuro de la citogenética en Costa Rica

This is an overview of the past, present and future of human Cytogenetics in Costa Rica. It started in 1965 at the University of Costa Rica where it has been developed slowly but steadily. There is only one overloaded clinical cytogenetic laboratory in the social security system. The tests currently performed are peripheral blood and blood marrow karyotypes, prenatal cytogenetic diagnosis (amniotic fluid and fetal blood) and less frequently skin biopsies. The task now is to standardize molecular cytogenetic techniques, we are actually working with PRINS in order to study submicroscopic subtelomeric rearrangements associated with mental retardation and other microdeletion syndromes as well.

Pregnancy outcomes of prenatally diagnosed Turner syndrome: a French multicenter retrospective study including a series of 975 cases.

The objectives of this study were to report pregnancy outcomes after prenatal diagnosis of Turner syndrome (TS) and to compare and assess termination of pregnancy rates during two periods. The intervals selected were before and after 1997 when multidisciplinary centers for prenatal diagnosis (MCPDs) were established in France. A database of 975 cases of TS diagnosed between 1980 and 2012 was created from 21 French cytogenetics laboratories. For each case, the karyotype indication, maternal age, year of prenatal testing, sampling procedure, karyotype, associated ultrasound findings, and outcomes were recorded. Karyotypes were mainly performed because of abnormal sonographic findings (84%). Before 1997, there were no changes in the rate of termination (90%) of affected fetuses. After 1997, the rate fell to 80%. This decrease was mainly observed in cases of mosaicism, incidental diagnosis, and in later gestations. US abnormalities were more likely to be associated with a full 45,X karyotype. There was an evolution in the way genetic counseling was performed following prenatal diagnosis of Turner syndrome that coincided with the opening of MCPDs in France. This resulted in a decrease in the rate of termination of affected fetuses.

ПРЕНАТАЛЬНЫЙ СКРИНИНГ ХРОМОСОМНЫХ АНОМАЛИЙ С ИСПОЛЬЗОВАНИЕМ ТРОЙНОГО МАРКЕРА

Метою представленого дослідження було вивчення ефективності скринінгу потрійних маркерів Каліфорнійської програми скринінгу, використовуючи дані з Реєстру хромосомних дефектів штату Каліфорнія (CCDR). У дослідження увійшло 752 686 жінок групи ризику, згідно скринінгу, з очікуваною датою пологів в період з липня 2005 року по кінець червня 2007-го. У схвалених державою пренатальних діагностичних центрах проводилося діагностичне спостереження за скринінг-позитивними жінками. Дані про результати візитів цих жінок були введені в Каліфорнійський реєстр хромосомних дефектів. Інші джерела Каліфорнійського реєстру хромосомних дефектів включають обов’язкову звітність усіх цитогенетичних лабораторій і лікарень, а також дані про результати вагітності з документацій пренатальних установ. Отримана чутливість скринінгу на синдром Дауна склала 77,4% (n = 1 217). Цей показник значно різнився залежно від методу оцінки гестаційного віку і віку матері. Чутливість скринінгу синдрому Дауна у жінок молодших 35 років і старшеих 35 років склала 62,4% і 94,0% відповідно. Чутливість скринінгу виявлення синдрому Дауна для вагітностей, діагностованих за допомогою УЗД, склала 81,3%, діагностованих за датою останньої менструації – 67,5%. Частота синдрому Тернера, трисомії 18, триплоїдії і трисомії 13 склала відповідно 79,4%, 82,5%, 98,1% і 36,0%. Скринінг- позитивна частота синдрому Дауна склала 5,4%. Тільки 49,5% жінок з синдромом Дауна у плода, які були скринінг-позитивними, пройшли амніоцентез. Серед жінок, у яких амніоцентез виявив синдром Дауна у плода, 61,4% вирішили перервати вагітність, 26,2% народили живих дітей, у 4,5% був викидень або мертвонародження і у 7,9% результат вагітності невідомий. Таким чином, ефективність цієї масштабної програми скринінгу з використанням потрійного маркеру перевищує звичайний прогностичний рівень виявлення синдрому Дауна. Методологія даного дослідження буде використовуватися для оцінки ефективності подальших удосконалень скринінгу.

Frequency and Types of Chromosomal Abnormalities in Turkish Women with Amenorrhea

Study ObjectiveTo estimate the frequency and the type of chromosomal abnormalities (CA) in patients with primary (PA) and secondary amenorrhea (SA). DesignThis retrospective study was comprised of patients had been referred to our laboratory between 1990 to 2008 and designed as original article. SettingMedical Faculty of Cukurova University in Turkey. ParticipantsChromosomal analysis was carried out on 393 patients with PA and SA that were referred to Cytogenetic laboratory of Medical Biology and Genetic Department, Faculty of Medicine, Çukurova University. InterventionsLymphocyte culturing depended karyotyping. Main Outcome MeasuresStandard lymphocyte culturing procedure and karyotyping was performed to all samples. ResultsPA and SA were identified in 393 patients. The karyotype was normal in 337 cases (85.8%) and abnormal in 56 (14.2%) patients. CAs were found in 54 (13.7%) and 2 (0.5%) of women with PA and SA, respectively. Females carrying rearrangements between autosomal and sex chromosomes were detected in 2% (8/393). The numerical abnormalities of the X chromosome were detected in 39.3% (22/56) (monosomy and mosaic). Structural abnormalities of the X and the other chromosomes were detected in 25.5% (13 of 56). Structural mosaicism of X chromosome was found in 5.4% (3 of 56). Male karyotype (46, XY) was found in 33.9% (19/56). The most frequently detected abnormality were X chromosome monosomies or mosaics. ConclusionsOur study revealed that some causes of amenorrhea could be due to CAs. Therefore, cytogenetic study should be important test in the evaluation of patients with PA or SA. The most common abnormality seen is 45,X karyotype (monosomy X/Turner Syndrome) and its variants.

Population-based characterization of the genetic landscape of chronic lymphocytic leukemia patients referred for cytogenetic testing in British Columbia, Canada: the role of provincial laboratory standardization

Detection of recurrent chromosome abnormalities by fluorescence in situ hybridization (FISH) is an essential component of care in chronic lymphocytic leukemia (CLL) patients. In the province of British Columbia (BC), Canada, population 4.6 million, CLL patients receive uniform evaluation and therapy with FISH testing performed in three jurisdictions. The aims of this study were to (i) validate CLL-FISH testing among the BC cytogenetic laboratories to ensure standardization of results and (ii) characterize population-level CLL-FISH abnormalities by pooling provincial data. From 2004 to 2011, 585 consecutive patients underwent pretreatment CLL-FISH testing at laboratory A (50.1%), laboratory B (32.3%), or laboratory C (17.6%). For validation purposes, 26 CLL-FISH abnormalities were tested by each laboratory's protocol, with 91% result concordance. Discordant results involved percent abnormalities at or near cutoff values; therefore, a 10% universal cutoff was established when pooling results. Applying the universal cutoff to the provincial cohort, CLL-FISH abnormalities were detected in 74.9%: 54.9% 13q-, 18.8% +12, 8.5% 11q-, and 7.7% 17p-. In this large population-based cohort of patients referred for CLL-FISH testing, frequencies of abnormalities detected by FISH analysis were highly consistent with those reported in single-institution and clinical trial populations. Provinces or districts that work together to care for CLL patients can effectively pool data with appropriate laboratory validation to ensure standardization of results.

Current status of prenatal diagnosis in Cuba: causes of low prevalence of Down syndrome.

To analyze trends in cytogenetic prenatal diagnosis in Cuba and to analyze possible causes leading to a low Down syndrome prevalence in a country where the triple test is not available. An analysis of the Cuban program in prenatal cytogenetic diagnosis from 1984 to 2012 was conducted. Results are described, with particular emphasis on indications, abnormal results, types of invasive procedures, and terminations of pregnancy. Cytogenetic prenatal diagnostic analyses (n = 75,095) were conducted; maternal age was the indication for 77.9% of the amniocenteses and chorionic villus samplings. The detection rate of chromosomally abnormal pregnancies was 2.3% for maternal age and increased to 8-9% for other indications. When a chromosomal abnormality was identified, 88.5% terminated the pregnancy. In 2002, the live birth prevalence of Down syndrome was 8.4 per 10,000 live births, and in 2012, 7 per 10,000. Prenatal diagnosis in Cuba has contributed to a significant reduction in chromosomal aberrations. The impact increased because of the demographic trends of the population, the high index of terminations of pregnancy, and the establishment of a network of cytogenetic laboratories throughout Cuba.

64P - Metastatic Breast Cancer: Getting the Facts from Peripheral Blood

Background: breast cancer is still a major challenge for oncologists worldwide, though giant steps made on orienting treatment, however, understanding tumor biology and cancer cell behavior is still at the beginning especially when we deal with an immortal, pluripotent cell.Objective: the study is aiming at isolating both circulating breast cancer cells and breast cancer stem cells, expanding them by cell culture and transforming them to paraffin embedded tissue like biopsy with a good yield upon immunohistochemistry.Patients and methods: the study was performed at al Bairouni university hospital and Kenj Cytogenetic Lab in Damascus (SYRIA). Blood samples were obtained from 100 chemonaive metastatic breast cancer patients. Samples were cultured in (DMEM) media, and after certain passages we obtained breast cancer stem cells CD44+/CD24- confirmed through passage in flowcytometer and PCR of protein product of CD44 surface protein. Then, obtained cells were subjected to immunohistochemistry for ER/PR/Her-2 after they were embedded in paraffin by means of Liquid based cytology.Results and discussion: we obtained good population of cells through expanding the circulating cancer cells and results of immunophenotyping were the same with the results obtained from the primary tumors (P 0.0001) except in 4 cases where Her-2 was positive on circulating cancer cells while it was negative on the primary.Conclusion: isolating tumor cells from circulation seems to be an efficient method in evaluating tumor characteristics and behavior. Our method is simple, cheap and informative; however, much work should be done to validate our results.Disclosure: All authors have declared no conflicts of interest. Background: breast cancer is still a major challenge for oncologists worldwide, though giant steps made on orienting treatment, however, understanding tumor biology and cancer cell behavior is still at the beginning especially when we deal with an immortal, pluripotent cell. Objective: the study is aiming at isolating both circulating breast cancer cells and breast cancer stem cells, expanding them by cell culture and transforming them to paraffin embedded tissue like biopsy with a good yield upon immunohistochemistry. Patients and methods: the study was performed at al Bairouni university hospital and Kenj Cytogenetic Lab in Damascus (SYRIA). Blood samples were obtained from 100 chemonaive metastatic breast cancer patients. Samples were cultured in (DMEM) media, and after certain passages we obtained breast cancer stem cells CD44+/CD24- confirmed through passage in flowcytometer and PCR of protein product of CD44 surface protein. Then, obtained cells were subjected to immunohistochemistry for ER/PR/Her-2 after they were embedded in paraffin by means of Liquid based cytology. Results and discussion: we obtained good population of cells through expanding the circulating cancer cells and results of immunophenotyping were the same with the results obtained from the primary tumors (P 0.0001) except in 4 cases where Her-2 was positive on circulating cancer cells while it was negative on the primary. Conclusion: isolating tumor cells from circulation seems to be an efficient method in evaluating tumor characteristics and behavior. Our method is simple, cheap and informative; however, much work should be done to validate our results. Disclosure: All authors have declared no conflicts of interest.

Establishing cytogenetic biodosimetry laboratory in Saudi Arabia and producing preliminary calibration curve of dicentric chromosomes as biomarker for medical dose estimation in response to radiation emergencies.

In cases of public or occupational radiation overexposure and eventual radiological accidents, it is important to provide dose assessment, medical triage, diagnoses and treatment to victims. Cytogenetic bio-dosimetry based on scoring of dicentric chromosomal aberrations assay (DCA) is the “gold standard” biotechnology technique for estimating medically relevant radiation doses. Under the auspices of the National Science, Technology and Innovation Plan in Saudi Arabia, we have set up a biodosimetry laboratory and produced a national standard dose–response calibration curve for DCA, pre-required to estimate the doses received. For this, the basic cytogenetic DCA technique needed to be established. Peripheral blood lymphocytes were collected from four healthy volunteers and irradiated with radiation doses between 0 and 5 Gy of 320 keV X-rays. Then, lymphocytes were PHA stimulated, Colcemid division arrested and stained cytogenetic slides were prepared. The Metafer4 system (MetaSystem) was used for automatic and manually assisted metaphase finding and scoring of dicentric chromosomes. Results were fit to the linear-quadratic dose–effect model according to the IAEA EPR-Biodosimetry-2011 report. The resulting manually assisted dose–response calibration curve (Y = 0.0017 + 0.026 × D + 0.081 × D2) was in the range of those described in other populations. Although the automated scoring over-and-under estimates DCA at low (<1 Gy) and high (>2 Gy) doses, respectively, it showed potential for use in triage mode to segregate between victims with potential risk to develop acute radiotoxicity syndromes. In conclusion, we have successfully established the first biodosimetry laboratory in the region and have produced a preliminary national dose–response calibration curve. The laboratory can now contribute to the national preparedness plan in response to eventual radiation emergencies in addition to providing information for decision makers and public health officials who assess the magnitude of public, medical, occupational and accidental radiation exposures.

Utilization of Magnetic-Activated Cell Sorting and High-Density Single Nucleotide Polymorphism Microarrays Improves Diagnostic Yield and Prognostic Value in Clinical Testing for Patients with Multiple Myeloma and Normal Routine Chromosome Study

Utilization of Magnetic-Activated Cell Sorting and High-Density Single Nucleotide Polymorphism Microarrays Improves Diagnostic Yield and Prognostic Value in Clinical Testing for Patients with Multiple Myeloma and Normal Routine Chromosome Study

Role of Dicentric Analysis in an Overarching Biodosimetry Strategy for Use Following a Nuclear Detonation in An Urban Environment

In the moments immediately following a nuclear detonation, casualties with a variety of injuries including trauma, burns, radiation exposure, and combined injuries would require immediate assistance. Accurate and timely radiation dose assessments, based on patient history and laboratory testing, are absolutely critical to support adequately the triage and treatment of those affected. This capability is also essential for ensuring the proper allocation of scarce resources and will support longitudinal evaluation of radiation-exposed individuals and populations. To maximize saving lives, casualties must be systematically triaged to determine what medical interventions are needed, the nature of those interventions, and who requires intervention immediately. In the National Strategy for Improving the Response and Recovery for an Improvised Nuclear Device (IND) Attack, the U.S. Department of Homeland Security recognized laboratory capacity for radiation biodosimetry as having a significant gap for performing mass radiation dose assessment. The anticipated demand for radiation biodosimetry exceeds its supply, and this gap is partly linked to the limited number and analytical complexity of laboratory methods for determining radiation doses within patients. The dicentric assay is a key component of a cytogenetic biodosimetry response asset, as it has the necessary sensitivity and specificity for assessing medically significant radiation doses. To address these shortfalls, the authors have developed a multimodal strategy to expand dicentric assay capacity. This strategy includes the development of an internet-based cytogenetics network that would address immediately the labor intensive burden of the dicentric chromosome assay by increasing the number of skilled personnel to conduct the analysis. An additional option that will require more time includes improving surge capabilities by combining resources available within the country's 150 clinical cytogenetics laboratories. Key to this intermediate term effort is the fact that geneticists and technicians may be experts in matters related to identifying chromosomal abnormalities related to genetic disorders, but they are not familiar with dosimetry for which training and retraining will be required. Finally, long-term options are presented to improve capacity focus on ways to automate parts of the dicentric chromosome assay method.

Isolated trisomy 2 in bone marrows of patients with suspected hematopoietic malignancies

Isolated trisomy 2 in hematopoietic malignancies is rare, having been reported in only eight cases. Of these cases, the majority are older males. The underlying hematologic malignancies range from myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). The molecular pathogenesis and prognostic significance of isolated trisomy 2 remains unknown. Herein, we report 11 cases of isolated trisomy 2 in hematologic disorders seen in the Mayo Clinic Cytogenetics laboratory from 1996-2012. The majority were older males between the ages of 63-93 years. The underlying bone marrow pathologic diagnoses ranged from no diagnostic features of malignancy to AML. Our data suggest that isolated trisomy 2 could represent an age-related phenomenon since all 11 cases were age 63 and over. It appears that isolated trisomy 2 harbors little prognostic significance and that, instead, the prognostic significance is driven by the underlying pathologic diagnosis. For example, whereas 3 of the cases with AML survived only 7-10 weeks post-bone marrow biopsy, 1 of the cases without diagnostic features of malignancy survived 10 additional years. Therefore, trisomy 2 as a sole abnormality should not be considered as definitive evidence for a myeloid neoplasm in the absence of diagnostic morphologic criteria.

Prenatal diagnosis of Prader–Willi syndrome and Angelman syndrome for fetuses with suspicious deletion of chromosomal region 15q11-q13

ObjectiveTo identify Prader–Willi syndrome (PWS) and Angelman syndrome (AS) among fetuses with suspicious deletion of the chromosomal region 15q11-q13. MethodsIn a retrospective study, data were assessed from fetuses missing chromosomal band 15q12 that underwent molecular diagnosis at the National Chen-Kung University Hospital, Tainan, Taiwan, between January 2001 and December 2012. Amniocytes were subjected to molecular testing, including fluorescence in situ hybridization (FISH) analysis, methylation-specific PCR (M-PCR), and methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA). ResultsDuring the 12-year study period, 26 041 amniocyte samples were analyzed at the study center and 27 (0.1%) were found to have a missing 15q12 band. A further 16 samples with a missing 15q12 band were received from other cytogenetic laboratories; as a result, 43 amniocyte samples lacking chromosomal band 15q12 underwent further molecular testing. Among these samples, 3 fetuses (7.0%) were found to have PWS (n=1) or AS (n=2). ConclusionA minority of cases with missing 15q12 had deletion of the PWS/AS critical region. This finding draws attention to the subtle structural rearrangements that occur on 15q11-q13 and provides useful information for prenatal diagnosis of PWS and AS.

The effects of biological and life-style factors on baseline frequencies of chromosome aberrations in human lymphocytes

Objective: This study investigated the influence of sex and ageing on chromosomal damage and the role of life-style habits on the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) of healthy Bosnian subjects. Materials and Methods: Peripheral blood samples were obtained from 100 healthy, unrelated individuals in Bosnia and Herzegovina during 2010 and 2011. Chromosome preparations were made by dropping and air drying and slides were stained with 10% Giemsa solution (pH 6.8). The cytogenetic analysis was carried out in a cytogenetic laboratory in the Department of Biology of the Faculty of Science in Sarajevo. The category of total structural CAs was sub classified as chromosome-type aberrations (CSAs) and chromatid-type aberrations (CTAs) while the category of total numerical CAs was sub classified as aneuploid and polyploid mitoses. All statistical analyses were carried out using Microsoft Excel 2010 (Microsoft Corporation) and the Windows Kwikstat Winks SDA 7.0.2 statistical software package (Texa Soft Cedar Hill, Texas). Results: Cytogenetic analysis revealed the average number of structural CAs was 2.84 and of numerical CAs was 9.56. There was a significant increase in the frequency of chromosome-type aberrations (1.92) compared with chromatid-type aberrations (CTAs) (0.92) and a significant increase in the frequency of aneuploid (8.83) compared with polyploid (0.73) mitoses. Significant positive correlations between age and CTAs in human PBLs were also demonstrated. Additional statistical analysis showed that ageing increase number of numerical CAs in lymphocytes of drinkers. The frequency of structural CAs of females exposed to radiation was significantly greater than in males. Analysis indicates the presence of a positive association between CAs and smoking in younger subjects but a negative correlation between aberrant cells frequencies and alcohol in older drinkers. Conclusion: The results of the study support the conclusion that sex and age, together with life-style habits of individuals as confounding factors can affect spontaneous frequency of CAs especially to CTAs in PBLs that are a biomarker of cancer risk.

Challenges in prenatal and pre-implantation genetic diagnosis studies.

Prenatal diagnosis (PND) of chromosomal anomalies by karyotyping and fluorescence in situ hybridization (FISH) is a routine procedure in many cytogenetic laboratories. However, PND of single gene disorders is available in only a few centres specializing in molecular genetic testing in India. Preimplantation genetic diagnosis (PGD) is a state-of the art technique involving biopsy of a single cell from a cleavage stage embryo obtained after intra-cytoplasmic sperm injection (ICSI). This is an additional step during in-vitro fertilization (IVF). The biopsied cell is then fixed on a slide in a critical step where the cytoplasm is removed to expose the nucleus. Single cells from many embryos of the couple are fixed and then screened for common aneuploidies by FISH using multicolour DNA probes. A maximum of 5 chromosomes can be checked in 1 hybridization. Hence the probes are stripped and slides are rehybridized in 2-4 rounds to study more chromosomes within a limited time frame. Only the chromosomally normal embryos are transferred or cryopreserved for a subsequent transfer. Karyotyping of couples with recurrent early miscarriages helps to identify the cause if the husband or wife is a carrier of a balanced translocation. PGD can help such couples to have a healthy baby earlier than by normal chance. Specific FISH probes have to be ordered especially for reciprocal translocations and a pre-PGD work up is important. This helped to identify an additional cryptic translocation between chromosomes 9 and 12 in a case from a neighbouring country, where the husband had inversion 12. Hence PGD included testing of chromosome 9 in this case. The wife has an ongoing pregnancy in the first attempt. Currently, ours is the only centre in India which offers PGD even for reciprocal/ Robertsonian translocations and microdeletions. We have had success with the birth of healthy babies. PGD for single gene disorders is yet in its infancy in our country. It involves genetic testing from DNA extracted from a single cell. Pre-PGD work up is more demanding, especially in cases where a HLA matched normal savior sibling is desired to cure a child affected with a hematological disorder such as Thalassemia.