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.

Chromosomes in the genomic age. Preserving cytogenomic competence of diagnostic genome laboratories.

Participation of clinical genetic laboratories in External Quality Assessment schemes (EQAs) is a powerful method to ascertain if any improvement or additional training is required in the diagnostic service. Here, we provide evidence from recent EQAs that the competence in recognizing and interpreting cytogenetic aberrations is variable and could impact patient management. We identify several trends that could affect cytogenomic competence. Firstly, as a result of the age distribution among clinical laboratory geneticists (CLGs) registered at the European Board of Medical Genetics, about 25-30% of those with experience in cytogenetics will retire during the next decade. At the same time, there are about twice as many molecular geneticists to cytogeneticists among the younger CLGs. Secondly, when surveying training programs for CLG, we observed that not all programs guarantee that candidates gather sufficient experience in clinical cytogenomics. Thirdly, we acknowledge that whole genome sequencing (WGS) has a great attraction to biomedical scientists that wish to enter a training program for CLG. This, with a larger number of positions available, makes a choice for specialization in molecular genetics logical. However, current WGS technology cannot provide a diagnosis in all cases. Understanding the etiology of chromosomal rearrangements is essential for appropriate follow-up and for ascertaining recurrence risks. We define the minimal knowledge a CLG should have about cytogenomics in a world dominated by WGS, and discuss how laboratory directors and boards of professional organizations in clinical genetics can uphold cytogenomic competence by providing adequate CLG training programs and attracting sufficient numbers of trainees.

Variability in Cytogenetic Testing for Multiple Myeloma: A Comprehensive Analysis From Across the United States.

PURPOSE: Multiple myeloma (MM) treatment has changed tremendously, with significant improvement in patient out-comes. One group with a suboptimal benefit is patients with high-risk cytogenetics, as tested by conventional karyotyping or fluorescence in situ hybridization (FISH). Methodology for these tests has been published, but not necessarily standardized. METHODS: We address variability in the testing and reporting methodology for MM cytogenetics in the United States using the ongoing African American Multiple Myeloma Study (AAMMS). We evaluated clinical and cytogenetic data from 1,221 patients (1,161 with conventional karyotyping and 976 with FISH) tested between 1998 and 2016 across 58 laboratories nationwide. RESULTS: Interlab and intralab variability was noted for the number of cells analyzed for karyotyping, with a significantly higher number of cells analyzed in patients in whom cytogenetics were normal (P 5.0025). For FISH testing, CD138-positive cell enrichment was used in 29.7% of patients and no enrichment in 50% of patients, whereas the remainder had unknown status. A significantly smaller number of cells was analyzed for patients in which CD138 cell enrichment was used compared with those without such enrichment (median, 50 v 200; P, .0001). A median of 7 loci probes (range, 1-16) were used for FISH testing across all laboratories, with variability in the loci probed even within a given laboratory. Chromosome 13-related abnormalities were the most frequently tested abnormality (n5956; 97.9%), and t(14;16) was the least frequently tested abnormality (n 5 119; 12.2%). CONCLUSIONS: We report significant variability in cytogenetic testing across the United States for MM, potentially leading to variability in risk stratification, with possible clinical implications and personalized treatment approaches.

Myelodysplastic syndrome: validation of flow cytometry multilineage score system.

OBJECTIVE: To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. METHODS: A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. RESULTS: Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. CONCLUSION: The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

How to Perform an Accurate Analysis of Metaphase I Chromosome Configurations in Autopolyploids of Arabidopsis thaliana.

During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).

Myelodysplastic syndrome: validation of flow cytometry multilineage score system / Síndrome mielodisplásica: validação de ficha de escore multilinhagem por citometria de fluxo

ABSTRACT Objective To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. Methods A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. Results Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. Conclusion The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

RESUMO Objetivo Validar ficha de escore multilinhagem correlacionando resultados obtidos de citometria de fluxo, citogenética, citomorfologia e histologia de amostras de pacientes com suspeita de síndrome mielodisplásica ou citopenias a esclarecer. Métodos Estudo retrospectivo de análise de dados laboratoriais de 49 pacientes com suspeita clínica de síndrome mielodisplásica ou citopenias a esclarecer realizado entre maio e setembro de 2017. Os critérios de inclusão foram a disponibilidade de resultados de citometria de fluxo e de, pelo menos, outra metodologia, entre morfologia, histologia, ou citogenética. Trinta e oito pacientes foram classificados como diagnosticados com síndromes mielodisplásicas enquanto 11 foram classificados como normais. Os pacientes foram avaliados utilizando sistemas de escore, escore de Ogata e ficha multilinhagem. Resultados Comparando as pontuações obtidas no escore de Ogata e na ficha multilinhagem, observou-se que, em quatro casos, o score de Ogata foi zero ou 1 ponto, enquanto, pela ficha multilinhagem, a pontuação foi superior a 3 pontos. Além disso, em 12 casos com escore de Ogata 2, a pontuação pela ficha multilinhagem foi superior a 3. Conclusão A ficha multilinhagem demonstrou ser mais eficaz na análise de displasia por avaliar as linhagens eritroide, monocítica, granulocítica e células precursoras, além dos parâmetros avaliados no escore de Ogata.

Acute Myeloid Leukemia, Version 3.2019, NCCN Clinical Practice Guidelines in Oncology.

Acute myeloid leukemia (AML) is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths due to leukemias in the United States. Recent advances have resulted in an expansion of treatment options for AML, especially concerning targeted therapies and low-intensity regimens. This portion of the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for AML focuses on the management of AML and provides recommendations on the workup, diagnostic evaluation and treatment options for younger (age <60 years) and older (age ≥60 years) adult patients.

Effects of platforms, size filter cutoffs, and targeted regions of cytogenomic microarray on detection of copy number variants and uniparental disomy in prenatal diagnosis: Results from 5026 pregnancies.

OBJECTIVE: We evaluated the effects of platforms, size filter cutoffs, and targeted regions of cytogenomic microarray (CMA) on the detection of copy number variants (CNVs) and uniparental disomy (UPD) in prenatal diagnosis. METHODS: Five thousand twenty-six consecutive prenatal specimens (>98% high-risk pregnancy) were studied by high-resolution CMA, with cutoffs of 50 kb for losses and 200 kb for gains in nontargeted regions and 20 kb for losses and 100 kb for gains in targeted regions. We assessed actual detection rates using the current assay as well as hypothetical detection rates using platforms with the same or lower resolution and smaller or larger cutoffs. RESULTS: The detection rate of our current assay was 11.2% (562 of 5026), including abnormal findings in 543 cases and likely pathogenic variants in 19. The hypothetical decrease in the overall detection of variants (excluding likely benign) and UPD ranged from 3.8% to 23.0%. For the subgroup of pathogenic and likely pathogenic CNVs < 1 Mb, the decrease of detection ranged from 2.7% to 24.3%. CONCLUSIONS: These findings underscore the significant effects of chosen CMA platform, as well as size filter cutoffs and targeted regions used in data analysis, on detection of CNVs and UPDs in a cohort of prenatal cases.

European guidelines for constitutional cytogenomic analysis.

With advancing technology and the consequent shift towards an increasing application of molecular genetic techniques (e.g., microarrays, next-generation sequencing) with the potential for higher resolution in specific contexts, as well as the application of combined testing strategies for the diagnosis of chromosomal disorders, it is crucial that cytogenetic/cytogenomic services keep up to date with technology and have documents that provide guidance in this constantly evolving scenario. These new guidelines therefore aim to provide an updated, practical and easily available document that will enable genetic laboratories to operate within acceptable standards and to maintain a quality service.

Advances in the role of cytogenetic analysis in the molecular diagnosis of B-cell lymphomas.

INTRODUCTION: Cytogenetic abnormalities represent essential determinants of diagnosis and prognosis in B-cell lymphomas. Their theranostic value is increasingly significant with the development of targeted therapies, in order to adapt the treatment at diagnosis as well as when relapse occurs. Areas covered: As the significance of these biomarkers is influenced by the technology used to detect them, an overview describing the strength and weakness of conventional and emerging technologies is provided. This review also updates the diverse cytogenetic abnormalities found in B-cell lymphomas, emphasizing their value in treatment decision. Expert commentary: Cytogenetics remains an essential analysis for the diagnostic work-up of lymphomas. As whole genome sequencing becomes more and more affordable routinely, the next challenge will be to recover all the information conveyed by conventional karyotype, including the analysis of the clonal architecture at the single cell level, in whole genome data.

A review of the challenge in measuring and standardizing BCR-ABL1.

Breakpoint cluster region-Abelson (BCR-ABL1) translocation is the characteristic sign of chronic myeloid leukemia (CML). The quantitation of BCR-ABL1 messenger RNA is requisite for patients with CML, and reverse-transcription real-time quantitative polymerase chain reaction (RQ-PCR) is the method used most extensively in testing laboratories worldwide. Nevertheless, substantial variation in RQ-PCR results from different laboratories makes interlaboratory comparability inconvincible owing to the lack of standardization. To facilitate interlaboratory comparative assessment and international standardization, an international scale (IS) for BCR-ABL1 was proposed. The laboratory-specific conversion factors derived from the IS can convert local different values to the IS without changing procedures. The standardization of BCR-ABL1 also includes the whole analytical process, so it is noteworthy to pay attention to the quality control before BCR-ABL1 quantitative analysis. More importantly, the World Health Organization has validated a first genetic reference panel which is limited to the manufacturers to produce and calibrate secondary reference reagents. Also, a certified reference plasmid, ERM-AD623, was internationally accepted. This article mainly focuses on BCR-ABL1 measurement and these standardization efforts in progress.

Investigation of the influence of calibration practices on cytogenetic laboratory performance for dose estimation.

PURPOSE: In the frame of the QA program of RENEB, an inter-laboratory comparison (ILC) of calibration sources used in biological dosimetry was achieved to investigate the influence of calibration practices and protocols on the results of the dose estimation performance as a first step to harmonization and standardization of dosimetry and irradiation practices in the European biological dosimetry network. MATERIALS AND METHODS: Delivered doses by irradiation facilities used by RENEB partners were determined with EPR/alanine dosimetry system. Dosimeters were irradiated in the same conditions as blood samples. A short survey was also performed to collect the information needed for the data analysis and evaluate the diversity of practices. RESULTS: For most of partners the deviation of delivered dose from the targeted dose remains below 10%. Deviations larger than 10% were observed for five facilities out of 21. Origins of the largest discrepancies were identified. Correction actions were evaluated as satisfactory. The re-evaluation of some ILC results for the fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC) assays has been performed leading to an improvement of the overall performances. CONCLUSIONS: This work has shown the importance of dosimetry in radiobiology studies and the needs of harmonization, standardization in irradiation and dosimetry practices and educational training for biologists using ionizing radiation.

Cytogenetics in the management of lymphomas and lymphoproliferative disorders in adults and children: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Non-Hodgkin's lymphomas and lymphoproliferative disorders include a high number of heterogeneous entities, described in the 2008 WHO classification. This classification reflects the crucial role of a multidisciplinary approach which integrates cytogenetic results both for the notion of clonality and for differential diagnosis between these entities. The prognostic impact of some cytogenetic abnormalities or genome complexity is also confirmed for many of these entities. Novel provisional entities have been described, such as BCLU (B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma) for which karyotype is critical to distinguish BCLU from Burkitt's lymphoma. The karyotype can be established from any tumour or liquid infiltrated by lymphoma cells. Recent adaptations of technics for cellular cultures according to the subtype of known (or suspected) lymphoma have significantly improved the percentage of informative karyotypes. Conventional karyotypes remain the best technical approach recommended for most of these subtypes. Interphase and/or metaphase FISH also represents a solid and rapid approach, because of the significant number of recurrent (sometimes specific) rearrangements of these entities. Next generation sequencing technologies contribute to enrich genomic data and substantially improve the understanding of oncogenic mechanisms underlying these lymphoid malignancies. Some molecular biomarkers are already part of the diagnostic process (for example, somatic mutation of MYD88 in Waldenström disease) thus reinforcing the essential principle of a multidisciplinary approach for the diagnosis of all the mature lymphoid malignancies.

Cytogenetics in the management of multiple myeloma: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Cytogenetics of multiple myeloma has evolved in recent years by the emergence of Interphasic fluorescence in situ hybridization (FISH) performed on sorted plasma cells detecting abnormalities independently of a proliferative and infiltrative index. Cytogenetic analysis plays a major part in the risk stratification of myeloma diagnosis due to prognostic impact of various cytogenetic abnormalities as well as to the association between emerging therapeutic approaches in MM. Thus, practice guidelines now recommend interphasic FISH or alternative molecular technics as the initial analysis for multiple myeloma. The Groupe francophone de cytogénétique hématologique (GFCH) proposes in this issue an update of managing multiple myeloma cytogenetics.

Cytogenetics in the management of children and adult acute lymphoblastic leukemia (ALL): an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Cytogenetic analyses (karyotype and, if necessary, appropriate complementary FISH analyses) are mandatory at diagnosis in acute lymphoblastic leukemia (ALL) as their results are taken into account in therapeutic protocols due to their diagnostic and prognostic values. In some cases, karyotype can be completed by other techniques (RT-PCR, RQ-PCR, DNA content, SNP-array, MLPA…) that can be equally or more informative than FISH. Here, we have tempted to establish guidelines concerning karyotype and FISH analyses according to the most recent data of the litterature which is reviewed here, completing the 2008 WHO classification with the recent new cytogenomic entities such as Ph-like ALL and indicating possible therapeutic implications.

Cytogenetics in the management of chronic lymphocytic leukemia: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

Acquired recurrent cytogenetic abnormalities are frequent in chronic lymphocytic leukaemia (CLL). They can be associated with good or poor prognostic factors, and also with gene mutations. Chromosomal abnormalities could be clonal or sub-clonal. Assessing the TP53 status (deletion/mutation) is currently mandatory before treating patients. The search for 11q deletion (ATM gene) is also recommended. Finally, the prognostic value of other chromosomal abnormalities including complex karyotype is still debated.

Cytogenetics in the management of acute myeloid leukemia: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

The karyotype is critical for the evaluation of acute myeloid leukemia (AML) at diagnosis. Cytogenetic abnormalities detected in AML are one of the most powerful independent prognostic factors. It impacts on the choice of treatment in clinical trials. All chromosomes can be targeted, common chromosomal abnormalities are recurrent and may be associated with a cytological well-defined type. In 40% of the cases, the karyotype is normal and must be associated with molecular biology studies that can refine the prognosis. The usefulness of the karyotype is more limited during the follow-up of the patient due to its limited sensitivity, but it is still useful in the clinical management of relapse. Since 2001, the WHO (World Health Organization) classification of hematological malignancies integrates cytogenetic data in the classification of AML. Karyotype is therefore mandatory in the diagnosis of AML.