No abstract

The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48h in-vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48h, resulting in an overall processing time of 72-96h for dose estimation.In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on xed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be speci c for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a ow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to approximately 2 hours. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid oxidation defect caused by a deficiency of the electron-transfer flavoprotein (ETF) or the electron-transfer flavoprotein dehydrogenase (ETFDH). There are three phenotypes of the disease, two neonatal forms and one late-onset. Previous studies have suggested that there is a phenotype–genotype correlation. We report on six patients from a single Bedouin tribe, five of whom were sequenced and found to be homozygous to the same variant in the ETFDH gene, with variable severity and age of presentation. The variant, NM_004453.3 (ETFDH): c.524G>A, p.(R175H), was previously recognized as pathogenic, although it has not been reported in the literature in a homozygous state before. R175H is located near the FAD binding site, likely affecting the affinity of FAD for EFT:QO. The single homozygous ETFDH pathogenic variant was found to be causing MADD in this cohort with an unexpectedly variable severity of presentation. The difference in severity could partly be explained by early diagnosis via newborn screening and early treatment with the FAD precursor riboflavin, highlighting the importance of early detection by newborn screening.

Introduction: Monitoring BCR-ABL fusion transcript levels in peripheral whole blood (WB) of patients on tyrosine kinase inhibitor (TKI) therapy using real-time quantitative PCR (RT-qPCR) is standard of care in the management of Chronic Myeloid Leukemia (CML). GeneXpert® BCR-ABL V2¥¥ or Xpert® BCR-ABL Ultra¥ (Ultra), a cartridge-based assay for use on the GeneXpert Instrument System, automates and standardizes the RT-qPCR process in less than 2 hours, using a lysate prepared from 4mL WB, resulting in an effective WB input volume of 600μL. There are, however, clinical situations where the total RNA isolated from high numbers of white blood cells (WBC) circulating in the patient's blood or in bone marrow (BM) can overload the Ultra cartridge and other quantitative BCR-ABL assays, requiring subsequent dilution of the sample to generate valid assay results. In this set of experiments, we sought to define the WBC input limits for Ultra that would predict cartridge overload, and thereby provide guidance regarding when to dilute the patient's sample in the presence of high WBC. Methods: Serial dilutions of WB specimens were tested in Ultra to determine the upper and lower WBC input limits corresponding to the valid ABL reference gene Ct cutoffs of 10 and 18, respectively. Sample prep procedures were developed to allow using 50μL or lower input volume from WB, BM, or the 1st sample lysate, with or without the WBC count (WBCC) information. BCR-ABL log% IS results from a subset of samples prepared and tested with various specimen input volumes in Ultra were compared in linear regression analyses with results from the Qiagen Ipsogen BCR-ABL1 Mbcr IS-MMR assay (IS-MMR). Results: The WBC input number of ~20 million cells/mL WB corresponded to the upper limit, and ~150K cells/mL WB corresponded to the lower limit, of the valid ABL Ct range for Ultra. For CML specimens with WBCCs <20 million cells/mL WB, the standard 4mL WB input yielded ABL Ct within acceptable limits. For specimens with WBCCs ≥20 million cells/mL, reaching as high as >500 million, sample prep procedures were developed to use 50μL WB, BM, or 1st lysate input volume. When 50μL was used but still yielded ABL Ct <10, a lower input volume (10μL or lower) can be used (Table 1). For situations in which the WBCC information is not available, but there is suspicion of high WBC yielding a very viscous 1st lysate that is hard to pipette, a 1st lysate treatment procedure for dilution was developed to allow for testing with 50μL or lower WB or BM input volume. These procedures were validated in Ultra by testing in CML specimens with various WB or BM input volume and showed high concordance (R2=0.98) when compared to the IS-MMR. Conclusions: In summary, sample prep procedures were developed in cases where high WBCC is known, or is suspected, or when repeat testing is needed for Invalids with ABL Ct <10, allowing the use of Ultra with various input sample volume for WB or BM in a wide variety of clinical situations. ¥ In vitro diagnostic medical device. May not be available in all countries. Not available in the United States. ¥¥ RUO; For research use only. Not for diagnostic use. Not reviewed by any regulatory body. Disclosures Day: Cepheid: Employment.

The dicentric chromosome assay (DCA), is considered the ‘gold standard’ for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48h in-vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24–48h, resulting in an overall processing time of 72–96h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to approximately 2 hours. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.