Transportation container for pre-processing cytogenetic assays in radiation accidents.

We report a shipping container that enables a disruptive logistics for cytogenetic biodosimetry for radiation countermeasures through pre-processing cell culture during transportation. The container showed precise temperature control (< 0.01 °C) with uniform sample temperature (< 0.1 °C) to meet the biodosimetry assay requirements. Using an existing insulated shipping box and long shelf life alkaline batteries makes it ideal for national stockpile. Dose curve of cytogenetic biodosimetry assay using the shipping container showed clear dose response and high linear correlation with the control dose curve using a laboratory incubator (Pearson's correlation coefficient: 0.992). The container's ability of pre-processing biological samples during transportation could have a significant impact on radiation countermeasure, as well as potential impacts in other applications such as biobanking, novel molecular or cell-based assays or therapies.

Is fluorescence in-situ hybridization sufficient in patients with myelodysplastic syndromes and insufficient cytogenetic testing?

Chromosome banding analysis (CBA) in myelodysplastic syndromes (MDS) remains the 'gold standard' for identification of chromosomal abnormalities, while interphase fluorescence in-situ hybridization (I-FISH) is mainly used to complement CBA. This study, retrospectively, evaluated CBA and I-FISH results in 600 patients with suspected MDS and determined the effect of CBA/FISH reallocation on IPSS-R. Our result demonstrated that in 7/586 (1.2%) patients with satisfactory karyotype, I-FISH provided additional information. In 25/453 (5.5%) of the patients with normal I-FISH, CBA detected chromosomal abnormalities, and in 68/147 (46%) of the patients with abnormal I-FISH, CBA detected additional chromosomal aberrations. When 5q- aberration was alone or accompanied by additional abnormalities by I-FISH, CBA revealed a complex karyotype (16/25;64%, 35/43;81%, respectively). Our results suggest that in cases of karyotype failure, if I-FISH is used alone, patients are at risk of being misclassified into the wrong cytogenetic risk groups and a repeat sample for CBA should be attempted.

Concise Review: Molecular Cytogenetics and Quality Control: Clinical Guardians for Pluripotent Stem Cells.

Now that induced pluripotent stem cell (iPSC)-based transplants have been performed in humans and organizations have begun producing clinical-grade iPSCs, it is imperative that strict quality control standards are agreed upon. This is essential as both ESCs and iPSCs have been shown to accumulate genomic aberrations during long-term culturing. These aberrations can include copy number variations, trisomy, amplifications of chromosomal regions, deletions of chromosomal regions, loss of heterozygosity, and epigenetic abnormalities. Moreover, although the differences between iPSCs and ESCs appear largely negligible when a high enough n number is used for comparison, the reprogramming process can generate further aberrations in iPSCs, including copy number variations and deletions in tumor-suppressor genes. If mutations or epigenetic signatures are present in parental cells, these can also be carried over into iPSCs. To maximize patient safety, we recommend a set of standards to be utilized when preparing iPSCs for clinical use. Reprogramming methods that do not involve genomic integration should be used. Cultured cells should be grown using feeder-free and serum-free systems to avoid animal contamination. Karyotyping, whole-genome sequencing, gene expression analyses, and standard sterility tests should all become routine quality control tests. Analysis of mitochondrial DNA integrity, whole-epigenome analyses, as well as single-cell genome sequencing of large cell populations may also prove beneficial. Furthermore, clinical-grade stem cells need to be produced under accepted regulatory good manufacturing process standards. The creation of haplobanks that provide major histocompatibility complex matching is also recommended to improve allogeneic stem cell engraftment. Stem Cells Translational Medicine 2018;7:867-875.

The Italian National External Quality Assessment Program in Cytogenetics: 4 years of activity (2013-2016) following the introduction of poor performance criteria.

BACKGROUND: Italian External Quality Assessment (IEQA) Program in Cytogenetics, established in 2001 by the Istituto Superiore di Sanità (ISS), covers both Constitutional and Oncohaematological diagnosis. In 2013, performance criteria were defined and adopted. In this paper, we present the data from the first 4 years of activity (2013-2016) following the introduction of performance criteria. METHODS: The enrollment is voluntary, fee-based and open to both public and private Italian laboratories. The scheme is annual and retrospective; a national panel of experts assess technical, analytical and interpretative performance. RESULTS: Overall, 95 distinct Italian laboratories participated in different Cytogenetics IEQA schemes over the 2013-2016 years and most of the laboratories took part in Constitutional diagnosis. General hospitals and local health centers represented 40% of the total participants and the percentage of laboratories from Northern Regions was more than 45% of total participants throughout the 4-year period. As regards the performance evaluation, on average, 11, 9 and 23% of participants were marked as poor performers in Prenatal, Postnatal and Oncohaematological schemes, respectively. With regard to critical errors, ISCN nomenclature in Prenatal and Postnatal schemes, and interpretation in Oncohaematological diagnosis, were identified as main issues. On the other hand, karyotype errors and inadequate analysis decreased strongly, over the 4 years, in Constitutional and Oncohaematological diagnosis, respectively. CONCLUSIONS: Our data show that the introduction of poor performance encourages laboratories to address critical issues, and the IEQA participation helps to improve quality in cytogenetic testing.

Factores pronósticos clínico-biológicos en pacientes con leucemia aguda mieloblástica en recaída / Clinical and biological prognostic factors in relapsed acute myeloid leukemia patients

Fundamentos y objetivo: La leucemia aguda mieloblástica (LAM) constituye la leucemia más frecuente en adultos. A pesar de los avances en el conocimiento de su patogenia, las tasas de curación no superan el 40%, siendo la recaída de la enfermedad la causa más frecuente de fallo de tratamiento. La recaída ocurre por fenómenos de evolución clonal. En este estudio analizamos los factores pronósticos clínicos y biológicos en pacientes adultos con LAM en recaída. Pacientes y métodos: Analizamos un total de 75 pacientes que presentaron recaída leucémica tras haber alcanzado la remisión completa. Se realizó un estudio inmunofenotípico mediante citometría de flujo y estudio citogenético mediante cariotipo convencional en muestras de médula ósea obtenidas en el momento del diagnóstico y de la recaída. Resultados: La supervivencia global (SG) de la serie fue del 3,7%±2,3, siendo la principal causa de muerte la progresión leucémica (83,3%). Los pacientes con recaídas precoces -antes de 12 meses- y aquellos con riesgo citogenético-molecular adverso presentaron SG significativamente inferiores. En el momento de la recaída el 52,5% de los pacientes mostraron cambios fenotípicos, y el 50%, cambios citogenéticos, sin observarse factores clínicos predictivos de dicha evolución clonal. La evolución clonal fenotípica o citogenética no mostró ningún impacto significativo en la SG. Conclusiones: Los pacientes con recaída de LAM presentan un pronóstico infausto, especialmente aquellos con recidiva precoz y riesgo citogenético-molecular adverso. La evolución clonal fenotípica y/o citogenética ocurre en la mitad de los casos sin factores clínicos predictivos ni impacto pronóstico (AU)

Background and objective: Acute myeloid leukemia (AML) is the most frequent type of acute leukemia in adults. Despite recent advances in the characterization of pathogenesis of AML, the cure rates are under 40%, being leukemia relapse the most common cause of treatment failure. Leukaemia relapse occurs due to clonal evolution or clonal escape. In this study, we aimed to analyze the clinical and biological factors influencing outcomes in patients with AML relapse. Patients and methods: We included a total of 75 AML patients who experienced leukaemia relapse after achieving complete remission. We performed complete immunophenotyping and conventional karyotyping in bone marrow aspirates obtained at diagnosis and at leukemia relapse. Results: Overall survival (OS) of the series was 3.7%±2.3, leukaemia progression being the most common cause of death. Patients relapsing before 12 months and those with adverse cytogenetic-molecular risk had statistically significant worse outcomes. A percentage of 52.5 of patients showed phenotypic changes and 50% cytogenetic changes at relapse. We did not find significant clinical factors predicting clonal evolution. The presence of clonal evolution at relapse did not have a significant impact on outcome. Conclusions: Patients with relapsed AML have a dismal prognosis, especially those with early relapse and adverse cytogenetic-molecular risk. Clonal evolution with phenotypic and cytogenetic changes occurred in half of the patients without predictive clinical factors or impact on outcome (AU)

CpG Oligonucleotide and Interleukin 2 stimulation enables higher cytogenetic abnormality detection rates than 12-o-tetradecanolyphorbol-13-acetate in Asian patients with B-cell chronic lymphocytic leukemia (B-CLL).

The present study was designed to compare abnormality detection rates using DSP30 + IL2 and 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Asian patients with B-CLL. Hematological specimens from 47 patients (29 newly diagnosed, 18 relapsed) were established as 72 h-DSP30 + IL2 and TPA cultures. Standard methods were employed to identify clonal aberrations by conventional cytogenetics (CC). The B-CLL fluorescence in situ hybridization (FISH) panel comprised ATM, CEP12, D13S25, and TP53 probes. DSP30 + IL2 cultures had a higher chromosomal abnormality detection rate (67 %) compared to TPA (44 %, p < 0.001). The mean number of analyzable metaphases and abnormal metaphases per slide was also higher (p < 0.005, p < 0.001, respectively). Culture success rate, percentage of complex karyotype, and percentage of non-clonal abnormal cell were not significantly different (p > 0.05). Thirteen cases with abnormalities were found exclusively in DSP30 + IL2 cultures compared to one found solely in TPA cultures. DSP30 + IL2 cultures were comparable to the FISH panel in detecting 11q-, +12 and 17p- but not 13q-. It also has a predilection for 11q- bearing leukemic cells compared to TPA. FISH had a higher abnormality detection rate (84.1 %) compared to CC (66.0 %) with borderline significance (p = 0.051), albeit limited by its coverage. In conclusion, DSP30 + IL2 showed a higher abnormality detection rate. However, FISH is indispensable to circumvent low mitotic indices and detect subtle abnormalities.

Potencial genotóxico del extracto oleoso de la semilla de Carapa guianensis Aublet para inducir anomalías en la morfología de la cabeza del espermatozoide / Genotoxic potencial of the Carapa guianensis Aublet seed oleaginous extract to induce abnormalities in sperm head morphology

Objetivo: La presente investigación se realizó con el objetivo de evaluar el potencial genotóxico del extracto oleoso de la semilla de Carapa guianensis para inducir anomalías en la morfología de la cabeza del espermatozoide en ratones machos Balb/c. Materiales y métodos: Se formaron 5 grupos experimentales: un grupo placebo (Tween 65 al 2%), 3 niveles de dosis del extracto (400, 1.000 y 2.000 mg/kg), administrados por vía oral durante 35 días, y por último un grupo control positivo tratado con ciclofosfamida (CF), en dosis de 50 mg/kg por vía intraperitoneal durante 5 días y 35 días de reposo después de la última administración con el mutágeno. Se distribuyeron 8 animales/grupo. Después de los 35 días de administración se eutanizaron por dislocación cervical y se extrajeron ambos epidídimos para ser procesados y evaluados según esta técnica citogenética. Resultados y discusión: Los resultados obtenidos entre controles y tratados con el extracto no difirieron en cuanto a la frecuencia de las diferentes anomalías de la cabeza del espermatozoide encontradas, y tampoco en la concentración espermática y de espermatozoides con gota citoplasmática. Sin embargo, sí difirieron controles y tratados contra el grupo tratado con CF, validando nuestros resultados. Se concluye que el extracto oleoso de la semilla de Carapa guianensis no posee potencialidades genotóxicas para inducir aumento en la frecuencia espontánea de la morfología de la cabeza del espermatozoide de ratones machos Balb/c (AU)

Objective: This study has aimed to evaluate the genotoxic potential of Carapa guianensis seed oil extract to induce sperm head abnormalities in Balb/c mice. Material and methods: Five experimental groups were formed: one placebo group (Tween 65, 2%), three extract dose levels (400, 1000 and 2000 mg/kg) administered orally for 35 days and one positive control group treated with cyclophosphamide (CP) at a dose of 50 mg/kg via intraperitoneally for 5 consecutive days and then 35 days of rest after the last administration with the mutagen. Were distributed 8 animals per group. The animals were euthanized by cervical dislocation 35 days after the administration and the epididymis was extracted to proceed with the sperm head morphology assay. Results and discussion: There were no significant differences in the results between the controls and those treated with the extract regarding frequency of the different sperm head anomalies or in the spermatic concentration and sperms with cytoplasmic drop. However, a difference was found between the controls and treated versus the CP treated group, validating our results. We have concluded that the oleaginous extract of Carapa guianensis seeds does not have genotoxic potentialities to induce an increase in the spontaneous frequency in the sperm head morphology of male Balb/c mice (AU)

Ejercicio físico y niveles urinarios de ácido úrico en el síndrome de Down / Physical exercise and urinary uric acid levels in Down’s syndrome

Antecedentes: Los individuos con síndrome de Down (SD) presentan niveles elevados de ácido úrico (AU). Objetivo: Evaluar la variación producida con la práctica de ejercicio físico en los niveles urinarios de AU en individuos con SD. Material y métodos: Se ha analizado a 29 individuos con SD de ambos sexos y edades de 4 a 52 años. Se analizaron 37 individuos sanos, sin trisomía 21 de ambos sexos y edades de 5-72 años (controles). Se utilizó el método de Duncan et al para determinar el AU. La creatinina (Cr) se determinó por el método de Jaffé, modificado por Varley y Gowenlock. Resultados: Los valores de AU urinario referenciados a Cr son significativamente mayores (p < 0,01) en individuos con SD que en controles (315 ± 123 mmol/mmol frente a 244 ± 83 mmol/mmol), y no varían significativamente ni con el sexo, ni con la edad. Sin embargo, tanto en el grupo control, como en el de SD aparece una correlación negativa entre el ratio AU/Cr y la edad hasta los 20 años, que se hace positiva a partir de esta edad. Nuestros resultados muestran una correlación más acentuada en personas con SD. El AU disminuye un 19% en SD y un 6,4% en controles cuando el deporte pasa de practicarse ocasionalmente a diariamente. Conclusiones: El AU urinario está aumentado en individuos con SD. El AU urinario no varía significativamente con el sexo. La práctica diaria de ejercicio físico con intensidad moderada reduce la excreción urinaria de AU en el SD (AU)

Background: Down’s syndrome (DS) individuals have elevated uric acid (UA) urinary levels. Objective: To evaluate urinary UA levels variation with physical exercise practice in DS individuals. Material and methods: We analysed 29 individuals with DS and 37 individuals without DS (control group) matched by age and sex. Urinary UA levels were determined by Duncan method. Creatinine (Cr) was assessed according to the spectrophotometric Jaffé method. Results: We reported that individuals with DS have significant elevated urinary UA levels compared to controls (315 ± 123 mmol/mmol vs 245 ± 84 mmol/mmol), and we did not observed any significant variation with respect to sex or age. However, up to 20 years a negative correlation between ratio UA/Cr and age was obtained. This correlation was positive starting from 20 years. According to our results this correlation is more accentuated in DS individuals. Urinary UA levels decrease 19.0% in DS individuals and 6.4% in controls when sport is practiced more than occasionally to daily. Conclusions: Urinary UA is increased in DS individuals. Urinary UA does not vary significantly according to sex. The daily practice of physical exercise of moderate intensity reduces the urinary excretion of UA in DS individuals (AU)

Edad materna: un factor de controversia en la trisomía 21 / Maternal age: a controversial factor in trisomy 21

Antecedentes: El síndrome de Down (SD) es la aneuploidía autosómica más frecuente en humanos. Se caracteriza por un fenotipo complejo que incluye rasgos faciales característicos y una apariencia esquelética; constituye la malformación congénita/síndrome de retraso mental más diagnosticado. Aunque la edad avanzada de la madre es un factor de riesgo bien establecido para la etiología del SD, sigue habiendo opiniones contrapuestas al respecto. Objetivo: El estudio se llevó a cabo para descubrir el efecto de la edad materna en la etiología de la trisomía 21. Material y métodos: Este estudio se realizó contemplando casos de SD de diferentes barrios del estado de Haryana (India). Los casos de SD estaban sujetos a un análisis morfológico y citogenético detallado. Resultados: En este estudio, más del 80% de los niños con SD habían nacido de madres jóvenes, de < 35 años, y menos del 20%, de madres de > 35 años. Los casos de SD nacidos de madres con edades inferiores a 30 años supusieron el 69,5%. La media de edad de la madre eran 29,5 años. El coeficiente de correlación parcial entre la edad de la madre y el número de casos de SD (manteniendo constante la edad del padre) se calculó como r = 0,315. Conclusión: El resultado de este estudio no es favorable al efecto de la edad materna avanzada en la incidencia de niños con SD. Podemos concluir que el riesgo de casos de SD no solamente se debe a la edad avanzada de la madre y que puede haber otros factores (genéticos y ambientales) que afecten la formación de un cigoto trisómico. Es necesario realizar más estudios para investigar los diferentes factores que regulan la segregación y la recombinación en humanos (AU)

Background: Down’s syndrome (DS) is the most common autosomal aneuploidy in human beings and is characterized by a complex phenotype including characteristic facial features, skeletal appearance and it is most commonly diagnosed congenital malformation/ mental retardation syndrome. Although advanced maternal age is a well established risk factor for the etiology of DS, controversy over it still continues. Objective: The study was carried out to find the effect of maternal age in the etiology of trisomy-21. Material and methods: Present study has been conducted on DS cases from various districts of Haryana State. DS cases were subjected to detailed morphological and cytogenetic analysis. Results: In the present study more than eighty percent of DS children were born to young mother’s of <35 years and less than twenty percent to mother’s age >35 years. DS cases born to mother’s of age less than 30 years were 69.5%. Mean age of mother was 29.5 years. Partial correlation coefficient between mother’s age and number of DS cases (keeping father age constant) was calculated as r = 0.315. Conclusion: Present study is not in favour of the effect of advanced maternal age on the occurrence of DS child. It can be concluded that risk of DS cases is not only due to the advanced maternal age and some others factors (genetic and environmental) may be involved in the formation of a trisomic zygote. Future studies are required to investigate the various factors that regulate the segregation & recombination in humans (AU)

Reacción en cadena de la polimerasa cuantitativa y cultivo largo: ¿el mejor método para el estudio citogenético de muestras de vellosidad corial? / Quantitative Polymerase Chain Reaction and long term culture: the best method for cytogenetic study of chorionic villus sampling?

El diagnóstico prenatal citogenético durante el primer trimestre de gestación se realiza a partir de biopsias de vellosidad corial. Para la obtención de metafases se utilizan dos métodos: el cultivo corto o semidirecto (STC) y cultivo largo (LTC). La principal ventaja del STC es que no presenta contaminación materna y la del LTC es que no hay descritos en la literatura falsos negativos. Se considera que la combinación de las dos técnicas (STC y LTC) es la estrategia diagnóstica más eficaz para este tipo de estudios. La técnica de PCR cuantitativa fluorescente (QF-PCR) permite evaluar las aneuploidías más frecuentemente implicadas en el diagnóstico prenatal en 24-48 horas en muestras de vellosidad corial. El objetivo de este trabajo es evaluar la combinación de QF-PCR y LTC como sustituto de las clásicas STC y LTC para el diagnóstico prenatal en muestras de vellosidad corial. Para ello presentamos nuestra experiencia en 900 muestras de vellosidad corial(AU)

First trimester cytogenetic prenatal diagnosis is performed on chorionic villus biopsies. Two methods are used to obtain metaphases: the short-term or semi-direct culture (STC) and long term culture (LTC). The main advantage of STC is that there is no risk of maternal contamination, and of LTC that no false-negative findings are described in the literature. It is considered that the combination of the two techniques (STC and LTC) is the most effective diagnostic strategy for this type of study. The technique of quantitative fluorescent PCR (QF-PCR) allows the evaluation of aneuploidy most frequently involved in prenatal diagnosis in 24-48 hours in chorionic villus samples. The aim of this study is to evaluate the combination of QF-PCR and LTC as a substitute for classical STC and LTC for prenatal diagnosis in chorionic villus samples. We present our experience in 900 chorionic villus samples(AU)

Standardization of fluorescence in situ hybridization studies on chronic lymphocytic leukemia (CLL) blood and marrow cells by the CLL Research Consortium.

Five laboratories in the Chronic Lymphocytic Leukemia (CLL) Research Consortium (CRC) investigated standardizing and pooling of fluorescence in situ hybridization (FISH) results as a collaborative research project. This investigation used fixed bone marrow and blood cells available from previous conventional cytogenetic or FISH studies in two pilot studies, a one-day workshop, and proficiency test. Multiple FISH probe strategies were used to detect 6q-, 11q-, +12, 13q-, 17p-, and IGH rearrangements. Ten specimens were studied by participants who used their own probes (pilot study 1). Of 312 FISH interpretations, 224 (72%) were true-negative, 74 (24%) true-positive, 6 (2%) false-negative, and 8 (3%) false-positive. In pilot study no. 2, each participant studied two specimens using identical FISH probe sets to control for variation due to probe sets and probe strategies. Of 80 FISH interpretations, no false interpretations were identified. At a subsequent workshop, discussions produced agreement on scoring criteria. The proficiency test that followed produced no false-negative results and 4% (3/68) false-positive interpretations. Interpretation disagreements among laboratories were primarily attributable to inadequate normal cutoffs, inconsistent scoring criteria, and the use of different FISH probe strategies. Collaborative organizations that use pooled FISH results may wish to impose more conservative empiric normal cutoff values or use an equivocal range between the normal cutoff and the abnormal reference range to eliminate false-positive interpretations. False-negative results will still occur, and would be expected in low-percentage positive cases; these would likely have less clinical significance than false positive results. Individual laboratories can help by closely following rigorous quality assurance guidelines to ensure accurate and consistent FISH studies in their clinical practice and research.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Central review of cytogenetics is necessary for cooperative group correlative and clinical studies of adult acute leukemia: the Cancer and Leukemia Group B experience.

The Cancer and Leukemia Group B has performed central review of karyotypes submitted by institutional cytogenetics laboratories from patients with acute myeloid (AML) and acute lymphoblastic (ALL) leukemia since 1986. We assessed the role of central karyotype review in maintaining accurate, high quality cytogenetic data for clinical and translational studies using two criteria: the proportion of karyotypes rejected (i.e. inadequate), and, among accepted (i.e. adequate) cases, the proportion of karyotypes whose interpretation was changed on central karyotype review. We compared the first four years during which central karyotype review was performed with a recent 4-year period and found that the proportion of rejected samples decreased significantly for both AML and ALL. However, during the latter period, central karyotype reviews still found 8% of AML and 16% of ALL karyotypes inadequate. Among adequate cases, the karyotype was revised in 26% of both AML and ALL samples. Some revisions resulted in changing the patients' assignment to particular World Health Organization diagnostic categories and/or moving patients from one prognostic group to another. Overall, when both data on rejection rates and data on karyotype revisions made in accepted cases were considered together, 32% of AML and 38% of ALL samples submitted were either rejected or revised on central karyotype review during the recent 4-year period. These data underscore the necessity of continued central karyotype review in multi-institutional cooperative group studies.

An Internet-based external quality assessment in cytogenetics that audits a laboratory's analytical and interpretative performance.

A novel approach to external quality assessment (EQA) using the Internet mimics the diagnostic situation so that multiple tests can be requested and EQA cases can be 'tailor made' to address a specific chromosome syndrome, disease, or clinical dilemma. The web-based EQA system was trialled on a large UK EQA scheme, UK NEQAS for Clinical Cytogenetics. It has also been used to implement a new Cytogenetics European Quality Assessment scheme, CEQA, set up with the intention of providing laboratories in countries without access to a local EQA scheme the opportunity of participation in EQA. Overall, Internet-based EQA allows for a varied EQA programme. Poor performance was detected in both CEQA and UK NEQAS constitutional EQA schemes and also in the UK NEQAS oncology EQA scheme. The Internet-based EQA overcomes submission delays due to international surface mail. There is also a reduction in administration and assessors' time compared to a retrospective EQA involving the submission of unique cases for EQA assessment, as participants analyse the same three Internet-based EQA cases simultaneously. Many EU27 (EU member states) laboratories still do not participate in their national EQA schemes, so until EQA participation becomes mandatory as a component of compulsory laboratory accreditation, the quality of laboratory diagnostic service is unpredictable.