Flow Fluorescence In Situ Hybridization Research Articles

Combined Flow-Fluorescence in situ hybridization to HHV-8 and EBV reveals the viral heterogeneity of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare B-cell non-Hodgkin lymphoma associated with Kaposi Sarcoma-associated herpesvirus (KSHV/HHV8) infection. Lymphoma cells are coinfected with Epstein-Barr virus (EBV) in 60-80% of cases. Tools allowing a reliable PEL diagnosis are lacking. This study reports PEL diagnosis in 4 patients using a Flow-Fluorescence in situ hybridization (FlowFISH) technique that allowed detection of differentially expressed EBV and HHV8 transcripts within the same sample, revealing viral heterogeneity of the disease. Moreover, infected cells exhibited variable expressions of CD19, CD38, CD40, and CD138. Therefore, FlowFISH is a promising tool to diagnose and characterize complex viral lymphoproliferations.

Identification of Adult Patients With Classical Dyskeratosis Congenita or Cryptic Telomere Biology Disorder by Telomere Length Screening Using Age-modified Criteria

Telomere biology disorders (TBD) result from premature telomere shortening due to pathogenic germline variants in telomere maintenance-associated genes. In adults, TBD are characterized by mono/oligosymptomatic clinical manifestations (cryptic TBD) contributing to severe underdiagnosis. We present a prospective multi-institutional cohort study where telomere length (TL) screening was performed in either newly diagnosed patients with aplastic anemia (AA) or if TBD was clinically suspected by the treating physician. TL of 262 samples was measured via flow-fluorescence in situ hybridization (FISH). TL was considered suspicious once below the 10th percentile of normal individuals (standard screening) or if below 6.5 kb in patients >40 years (extended screening). In cases with shortened TL, next generation sequencing (NGS) for TBD-associated genes was performed. The patients referred fell into 6 different screening categories: (1) AA/paroxysmal nocturnal hemoglobinuria, (2) unexplained cytopenia, (3) dyskeratosis congenita, (4) myelodysplastic syndrome/acute myeloid leukemia, (5) interstitial lung disease, and (6) others. Overall, TL was found to be shortened in 120 patients (n = 86 standard and n = 34 extended screening). In 17 of the 76 (22.4%) standard patients with sufficient material for NGS, a pathogenic/likely pathogenic TBD-associated gene variant was identified. Variants of uncertain significance were detected in 17 of 76 (22.4%) standard and 6 of 29 (20.7%) extended screened patients. Expectedly, mutations were mainly found in TERT and TERC. In conclusion, TL measured by flow-FISH represents a powerful functional in vivo screening for an underlying TBD and should be performed in every newly diagnosed patient with AA as well as other patients with clinical suspicion for an underlying TBD in both children and adults.

High-Grade B-Cell Lymphoma With Malignant Effusions as the Initial Presentation.

Malignant effusion is usually caused by metastatic carcinoma. Malignant lymphoma is often not included as a top differential diagnosis of malignant effusion. Here, we describe 3 cases of young female patients with no significant past medical history who presented with fluid overload and were diagnosed with high-grade B-cell lymphoma (HGBL). We conducted histopathologic examination and immunophenotypic and cytogenetic analyses on three cases using immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), and karyotyping. We also included patients' clinical and radiological findings in our case reports. Histologic examination of the effusion samples showed numerous intermediate to large lymphoma cells with irregular nuclear contours and fine chromatin. The lymphoma cells were positive for CD10, CD20, BCL2, BCL6, and PAX5 and negative for CD34, cyclin D1, HHV-8, and TdT. In situ hybridization for Epstein-Barr virus (EBV)-encoded small RNAs was negative. The proliferation index by Ki-67 stain was more than 80%. Flow cytometry showed CD10-positive B cells with monotypic immunoglobulin light chain expression. Fluorescence in situ hybridization analysis demonstrated MYC, BCL2, or BCL6 rearrangements. These 3 patients were diagnosed as having HGBL with double-/triple-hit rearrangements. Despite receiving aggressive chemotherapy, all 3 patients had a dismal clinical course, with 2 patients dying less than 2 years after initial diagnosis. High-grade B-cell lymphoma should be considered in the differential diagnoses of malignant effusions. Flow cytometric and FISH analyses of the body fluid specimens are essential to reach an accurate and timely diagnosis.

Impact of inorganic iron and haem on the human gut microbiota; An in vitro batch-culture approach.

Although iron is an essential nutrient for humans, as well as for almost all other organisms, it is poorly absorbed (~15%) from the diet such that most passes through the upper gut into the large intestine. The colonic microbiota is thus exposed to, and potentially influenced by, such residual iron which could have an impact on human health. The aim of the research described here is to determine how the major forms of dietary iron (inorganic iron and haem) influence metabolic activity and composition of the human gut microbiota by utilizing an in vitro parallel, pH-controlled anaerobic batch culture approach. Controlled iron provision was enabled by the design of a 'modified' low-iron gut-model medium whereby background iron content was reduced from 28 to 5 μM. Thus, the impact of both low and high levels of inorganic and haem iron (18-180 μM and 7.7-77 μM, respectively) could be explored. Gut-microbiota composition was determined using next generation sequencing (NGS) based community profiling (16S rRNA gene sequencing) and flow-fluorescent in situ hybridization (FISH). Metabolic-end products (organic acids) were quantified using gas chromatography (GC) and iron incorporation was estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that differences in iron regime induced significant changes in microbiota composition when low (0.1% w/v) fecal inoculation levels were employed. An increase in haem levels from 7.7 to 77 μM (standard levels employed in gut culture studies) resulted in reduced microbial diversity, a significant increase in Enterobacteriaceae and lower short chain fatty acid (SCFA) production. These effects were countered when 18 μM inorganic iron was also included into the growth medium. The results therefore suggest that high-dietary haem may have a detrimental effect on health since the resulting changes in microbiota composition and SCFA production are indicators of an unhealthy gut. The results also demonstrate that employing a low inoculum together with a low-iron gut-model medium facilitated in vitro investigation of the relationship between iron and the gut microbiota.

High-Grade B-Cell Lymphoma with Effusion as the Initial presentation

Abstract Introduction/Objective Malignant effusion is usually caused by metastatic carcinoma. Lymphoma is often not included in the differential diagnoses. Here, we describe two cases of young females with no significant past medical history, presenting with fluid overload. Methods/Case Report The first case is a 41-year-old female with abdominal ascites and pleural effusion. Microscopic examination of the ascitic fluid showed intermediate to large lymphoid cells with irregular nuclear contours and fine chromatin. Flow cytometry showed CD10-positive lambda restricted B-cells. She had retropectoral mass biopsy showing sheets of intermediate to large-sized lymphoma cells with abundant apoptotic bodies and necrosis. The lymphoma cells are positive for CD20, CD10, BCL-2, BCL-6, and C-MYC (~50%,) while negative for TdT and cyclin D1. Proliferative index by Ki-67 stain is ~80%). Fluorescence in situ hybridization (FISH) analysis demonstrated rearrangements of BCL2/18q (80%) and MYC/8q (82%). The patient had a diagnosis of high-grade B-cell lymphoma with MYC and BCL-2 gene rearrangements. The second case is a 43-year-old woman with abdominal pain. She was diagnosed with acute cholecystitis and underwent cholecystectomy. Peritoneal fluid showed numerous neoplastic cells with irregular nuclei, prominent nucleoli, and cytoplasmic vacuoles. Flow cytometry demonstrated CD10-positive B-cells with lambda restriction. The cells were positive for CD10, CD20, BCL-2, BCL-6, and PAX5, and negative for CD34, cyclin D1, HHV-8, and TDT. EBER ISH was negative. The proliferation index by Ki67 stain was >90%. FISH studies showed MYC, BCL2 and BCL6 gene arrangements. Cytogenetic analysis revealed a complex karyotype. A diagnosis of HGBL with triple-hit rearrangements was rendered. Despite receiving aggressive chemotherapy, the patient expired 8.5 months after the diagnosis. Results (if a Case Study enter NA) NA. Conclusion HGBL is an aggressive and often stage IV disease. It should be considered in the differential diagnoses of malignant effusions. Flow cytometry and FISH analysis of the body fluid specimen are essential to reach an accurate diagnosis in a timely manner.

Characterization of tree shrew telomeres and telomerase

The use of tree shrews as experimental animals for biomedical research is a new practice. Several recent studies suggest that tree shrews are suitable for studying cancers, including breast cancer, glioblastoma, lung cancer, and hepatocellular carcinoma. However, the telomeres and the telomerase of tree shrews have not been studied to date. Here, we characterize telomeres and telomerase in tree shrews. The telomere length of tree shrews is approximately 23 kb, which is longer than that of primates and shorter than that of mice, and it is extended in breast tumor tissues according to Southern blot and flow-fluorescence in situ hybridization (FISH) analyses. Tree shrew spleen, bone marrow, testis, ovary, and uterus show high telomerase activities, which are increased in breast tumor tissues by telomeric repeat amplification protocol assays. The telomere length becomes shorter, and telomerase activity decreases with age. The tree shrew TERT and TERC are more highly similar to primates than to rodents. These findings lay a solid foundation for using tree shrews to study aging and cancers.

Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.

Doxorubicin-induced senescence through NF-κB affected by the age of mouse mesenchymal stem cells.

The senescence is proposed as a defense mechanism against many anticancer drugs. This complication is marked by differences in cell appearance and inner structures underlying the impairment in function. In this experiment, doxorubicin-induced senescence was assessed in mesenchymal stem cells (MSCs) isolated from the bone marrow of different-aged Balb/c mice (1, 8, and 16 months old). In addition, doxorubicin kinetics in culture medium were investigated to compare the drug absorption rate by different-aged MSCs. Several methods were exerted including Sandwich ELISA for NF-κB activation, propidium iodide staining for cell cycle analysis, Flow-fluorescent in-situ hybridization (Flow-FISH) assay for telomere length measurement, and specific staining for evaluation of β-galactosidase. Determination of doxorubicin in a medium was performed by high-performance liquid chromatography technique. Following doxorubicin exposure, cells underwent substantial telomere shortening, cell cycle arresting in G2 phase, and increased β-galactosidase activity. Interestingly, the enhanced level of NF-κB was observed in all age groups. The highest and lowest sensitivity to telomere shortening attributed to 1- and 8-month-old MSCs, respectively. In consistent with Flow-FISH results, the β-galactosidase activity was higher in young-aged MSCs after treatment. Statistical analysis indicated a correlation between the reduction of telomere length and cessation in G2 phase. Regarding the obtained kinetics equations, the rate of doxorubicin absorption by all aged MSCs followed the same trend. In conclusion, the changing of some elements involved in doxorubicin-induced senescence can be affected by the age of the cells significantly in young MSCs than two other age groups. Hereupon, these changing patterns can open new insights to develop anticancer therapeutic strategies.

Microfluidic enrichment of plasma cells improves treatment of multiple myeloma.

Cytogenetic alterations form the basis for risk stratification for multiple myeloma (MM) and guide the selection of therapy; however, current pathology assays performed on bone marrow samples can produce false‐negatives due to the unpredictable distribution and rarity of MM cells. Here, we report on a microfluidic device used to facilitate CD45 depletion to enhance the detection of cytogenetic alterations in plasma cells (PCs). Bone marrow samples from 48 patients with MM were each divided into two aliquots. One aliquot was subjected to classic flow cytometry and fluorescent in situ hybridization (FISH). The other first went through CD45+ cell depletion, further enriched by microfluidic size selection. The enriched samples were then analyzed using flow cytometry and FISH and compared to those analyzed using the classic method only. Unlike the traditional method, the microfluidic device removed the CD45+ leukocytes and specifically selected PCs from the remaining white blood cells. Therefore, the microfluidic method (MF‐CD45‐TACs) significantly increased the percentage of CD38+/CD138+ cells to 37.7 ± 20.4% (P < 0.001) from 10.3 ± 8.5% in bone marrow. After the MF‐CD45‐TAC enrichment, the detection rate of IgH rearrangement, del(13q14), del(17p), and 1q21 gains, rose to 56.3% (P < 0.001), 37.5% (P < 0.001), 22.9% (P < 0.001), and 41.7% (P = 0.001), respectively; all rates of detection were significantly increased compared to the classically analyzed samples. In this clinical trial, this microfluidic‐assisted assay provided a precise detection of cytogenetic alterations in PCs and improved clinical outcomes.

Biological Age and Circulating Progenitor Cell Levels as Predictors Heart Disease Events

Identifying patients with stable coronary artery disease (CAD) at high risk for future heart attacks or death remains a challenge for all cardiologists. The study entitled Telomere Shortening, Regenerative Capacity, and Cardiovascular Outcomes in this issue has shed new light in the field.1 Hammadah et al1 focused on the relationship between circulating CD34+ cell levels, telomere length, and cardiovascular outcomes in subjects with stable CAD. Their study yielded 2 novel findings: (1) 10% shorter telomeres correlated with 5% lower levels of circulating CD34+ cells and (2) both short leukocyte telomere length (LTL) and low CD34+ cell levels independently predict adverse cardiovascular disease (CVD) outcomes (death, myocardial infarction [MI], coronary revascularization, or cerebrovascular events). Measurements of LTL and circulating CD34+ cells may facilitate risk stratification of patients with stable CAD distinct from traditional CVD risk factors. Prior studies reported associations between shortened LTL and CVD,2 and postulated that decreased regenerative capacity may be a primary cause of CVD events. The study by Hammadah et al1 is the first and largest study to test the hypothesis that LTL may be linked to the levels of CD34+ cells as a surrogate for regenerative capacity, and correlate these measures with CVD events. Article, see p 1130 Telomeres prevent chromosome ends from being recognized as a DNA break and activating DNA damage response pathways …

Clinical Spectrum of Germline Mutations with Predisposition to Myeloid Neoplasms- 2016 World Health Organization Classification Update

▪Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision.Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision.Results: 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified.Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1).Conclusion: The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding.

A rare case of acute promyelocytic leukemia with focal bone marrow involvement presenting as a paraspinal myeloid sarcoma

Acute promyelocytic leukemia (APL) accounts for 5–8% of acute myeloid leukemias (AMLs). Myeloid sarcomas (MS) are rare extramedullary tumors consisting of immature myeloid cells. These tumors may develop de novo or concurrently with AML and may precede or coincide with the occurrence of AML. The occurrence of MS in an APL patient is a rare clinical event. In these cases, it usually occurs as a sign of relapse. Focal bone marrow involvement by APL is also rare; the majority of cases of APL demonstrate a diffusely hypercellular bone marrow throughout the axial skeleton due to expansion of neoplastic blasts/promyelocytes. We describe a 23-year-old female who presented with gait problems, including multiple falling episodes, and lower extremity weakness. An MRI demonstrated a paraspinal soft tissue mass. Histopathologic analysis demonstrated a myeloid sarcoma expressing CD45, myeloperoxidase, CD117, BCL2, focal weak CD34, and weak CD99 and negative for CD30, TdT, CD20, CD3, and CD10. The bone marrow aspirate demonstrated blasts/promyelocytes accounting for approximately 26% of the total bone marrow aspirate cellularity. The bone marrow biopsy and clot section demonstrated normocellularity with a few blast/promyelocyte foci on the clot section accounting for approximately 5% of total bone marrow cellularity. These foci expressed MPO and CD117 and were negative for CD34. Flow cytometry demonstrated blasts accounting for approximately 31% of all CD45+ events expressing MPO, CD33, CD13, CD117, CD64, CD2, CD38, and CD71 and negative for CD34, HLA-DR, CD11b, CD14, CD15, CD16, CD19, CD22, CD10, CD56, CD61, and surface and cytoplasmic CD3. Fluorescence in situ hybridization (FISH) analysis was positive for the t(15;17)(q22;q12) promyelocytic leukemia/retinoic acid receptor alpha (PML/RARA) translocation. The patient was induced with arsenic trioxide and subsequently underwent consolidation chemotherapy with daunorubicin and all-trans-retinoic acid. Her post-chemotherapy bone marrow biopsy was negative for residual disease. Flow cytometry and FISH analysis were also negative. This is a rare case of APL involving both the initial presentation as myeloid sarcoma and focal bone marrow involvement.

A Rare Case of Acute Promyelocytic Leukemia With Focal Bone Marrow Involvement Presenting as a Paraspinal Myeloid Sarcoma

Acute promyelocytic leukemia (APL) accounts for 5–8% of acute myeloid leukemias (AMLs). Myeloid sarcomas (MS) are rare extramedullary tumors consisting of immature myeloid cells. These tumors may develop de novo or concurrently with AML and may precede or coincide with the occurrence of AML. The occurrence of MS in an APL patient is a rare clinical event. In these cases, it usually occurs as a sign of relapse. Focal bone marrow involvement by APL is also rare; the majority of cases of APL demonstrate a diffusely hypercellular bone marrow throughout the axial skeleton due to expansion of neoplastic blasts/promyelocytes. We describe a 23-year-old female who presented with gait problems, including multiple falling episodes, and lower extremity weakness. An MRI demonstrated a paraspinal soft tissue mass. Histopathologic analysis demonstrated a myeloid sarcoma expressing CD45, myeloperoxidase, CD117, BCL2, focal weak CD34, and weak CD99 and negative for CD30, TdT, CD20, CD3, and CD10. The bone marrow aspirate demonstrated blasts/promyelocytes accounting for approximately 26% of the total bone marrow aspirate cellularity. The bone marrow biopsy and clot section demonstrated normocellularity with a few blast/promyelocyte foci on the clot section accounting for approximately 5% of total bone marrow cellularity. These foci expressed MPO and CD117 and were negative for CD34. Flow cytometry demonstrated blasts accounting for approximately 31% of all CD45+ events expressing MPO, CD33, CD13, CD117, CD64, CD2, CD38, and CD71 and negative for CD34, HLA-DR, CD11b, CD14, CD15, CD16, CD19, CD22, CD10, CD56, CD61, and surface and cytoplasmic CD3. Fluorescence in situ hybridization (FISH) analysis was positive for the t(15;17)(q22;q12) promyelocytic leukemia/retinoic acid receptor alpha (PML/RARA) translocation. The patient was induced with arsenic trioxide and subsequently underwent consolidation chemotherapy with daunorubicin and all-trans-retinoic acid. Her post-chemotherapy bone marrow biopsy was negative for residual disease. Flow cytometry and FISH analysis were also negative. This is a rare case of APL involving both the initial presentation as myeloid sarcoma and focal bone marrow involvement.

Telomere length of peripheral lymphocytes in patients with immuno-related pancytopenia

To investigate the changes of relative telomere length (RTL) of peripheral blood (PB) CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺T lymphocytes, CD19⁺B lymphocytes and bone marrow (BM) CD34⁺ cells and its association with disease severity in untreated patients with immuno-related pancytopenia (IRP). The PB CD3⁺ , CD3⁺ CD4⁺ , CD3⁺ CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and BM CD34⁺ cells were purified by magnetic activated cell sorting (MACS), and RTL were measured with flow-fluorescence in situ hybridization (FLOW-FISH). The RTL of CD3⁺, CD3⁺CD4⁺ , and CD3⁺CD8⁺T lymphocytes in untreated IRP patients were (27.754 ± 16.323)%, (7.526 ± 3.745)% and (25.854 ± 14.789)%, respectivly, which were significantly shorter than those in healthy-controls (54.555 ± 19.782)%, (12.096 ± 2.805)%, and (38.367 ± 4.626)% (P<0.05). The RTL of CD19⁺ lymphocytes in untreated IRP patients was (22.136 ± 16.142)%, which was significantly shorter than that in healthy controls (42.846 ± 16.353)% (P<0.01). There was no significant difference of BM CD34⁺ cells RTL between the untreated IRP patients (22.528 ± 21.601)% and the healthy controls (23.936 ± 19.822)% (P>0.05). There were significantly positive correlations between the RTL of B lymphocytes and the count of white blood cell (r=0.706, P=0.015). There were negative correlations between RTL of B lymphocytes and the clinical symptoms (r=-0.613, P=0.045) and positive correlations with therapeutic effect (r=0.775, P=0.005). The shorter RTL of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ lymphocytes, and the normal RTL of BM CD34⁺ cells in untreated IRP patients were identified, which might imply that IRP is a type of acquired autoimmune diseases.

Identification of Markov Chains From Distributional Measurements and Applications to Systems Biology

In this paper, we are concerned with doing system identification for biological systems using time series distributional measurements and Markov chain models. By distributional measurements, we mean measurements provided by assays such as flow cytometry or FISH (fluorescence in situ hybridization) that allow us to make the same single cell measurements over a number of different cells. We focus here on the problem of estimating the transition probabilities of a Markov chain from distributional measurements. We set this problem up using Bayes' rule and make simplifying assumptions to reduce the problem to a non-convex optimization problem over finitely many variables. We propose methods for locally solving this non-convex optimization problem. For a special case, we discuss necessary and sufficient conditions for the Markov chain to be identifiable. Finally, we demonstrate our procedure on instructive toy examples as well as on simulated stochastic data for a genetic toggle switch. [Gardner et al. (2000)].

Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses

Shoot organogenesis was induced from 2- and 6-week-old callus derived from the leaves of Arabidopsis thaliana ecotype Columbia (2n = 10). Regenerated plants were evaluated for chromosomal variations by means of flow cytometry and fluorescent in situ hybridization (FISH). Flow cytometric measurements revealed the occurrence of diploid, tetraploid, and octoploid plants among the regenerants of 2-week-old calli, whereas only diploid and tetraploid plants were regenerated from the 6-week-old calli. Chromosome counting showed that plants developed from the 2-week-old calli exhibited mixoploidy and a high frequency of aneuploid cells. These plants were infertile and displayed altered morphology. FISH with 5S and 25S rDNA probes allowed to detect some structural chromosomal rearrangements in regenerated plants. Along with cells which exhibited correct localisation of rDNA loci, also cells bearing chromosomal translocations, deletions or duplications were found. The type of structural aberrations varied between diploid and tetraploid regenerants.

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

Three novel truncating TINF2 mutations causing severe dyskeratosis congenita in early childhood

Dyskeratosis congenita (DC) is a telomere biology disorder characterized by a mucocutaneous triad, aplastic anemia, and predisposition to cancer. Mutations in a narrow segment of TINF2 exon 6 have been recognized to cause often-severe DC that is either sporadic or autosomal dominant. We describe three children with very early presentations of DC, including one with the severe variant known as Revesz syndrome. Although most TINF2 mutations reported to date are missense changes, each of our patients carried a novel heterozygous nonsense or frameshift mutation, revealing a new 5' boundary to the affected gene segment in patients with DC. Examination of patient-derived lymphoblastoid cell lines revealed stable expression of the predicted truncated TIN2 proteins. In co-immunoprecipitation assays, the ability of a truncation mutant to interact with TRF1 was severely impaired, whereas the ability of the most common DC-associated mutant was much less affected. This suggests that the disruption of TIN2-TRF1 interaction may contribute to the severe clinical phenotype observed in the context of the TIN2 truncation mutation, but is unlikely to be the primary cause of telomere shortening associated with the more prevalent TIN2 missense mutations. Telomere flow-fluorescent in situ hybridization (FISH) analysis of one pedigree showed the dramatic effect a de novo nonsense TINF2 mutation had on telomere length in early development. These cases underscore the severe manifestations of truncating TINF2 mutations.

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non-DNA binding genotoxins (e.g., aneugens), unlike DNA-binding genotoxins, are theoretically expected to show thresholded concentration-effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose-response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non-linear dose-dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg(-1), respectively.

Correlation of Telomere Length in Blood, Buccal Cells, and Fibroblasts From Patients with Inherited Bone Marrow Failure Syndromes.

Abstract 1083Poster Board I-105 IntroductionPatients with dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS), have defects in telomere biology. Measurement of telomere length (TL) by flow fluorescence in situ hybridization (FISH) in white blood cell (WBC) subsets is a very important diagnostic tool in DC. Quantitative PCR (Q-PCR) is a high-throughput method of TL measurement that is currently used in large epidemiologic studies. It measures telomere length as a ratio of TTAGGG repeat copy number to a single copy gene number (T/S), and is often used on DNA derived from blood or buccal cells. There are limited data on tissue-specific correlations of TL and on the comparability of studies using different methods. In order to better understand tissue TL variability, we evaluated intra-individual correlations of DNA extracted from frozen blood, fibroblasts, and buccal cells by Q-PCR; and flow-FISH TL in WBC subsets. Patients and MethodsWe studied 21 patients: 5 Diamond-Blackfan Anemia (DBA), 6 DC, 5 Fanconi anemia (FA), and 4 Shwachman-Diamond Syndrome (SDS), enrolled in the National Cancer Institute's IBMFS study who contributed blood, buccal cells, and fibroblasts, and also had WBC flow-FISH TL measured. Genomic DNA was extracted from either whole blood (n=4) or the WBC pellet remaining after ficoll separation (n=17, primarily granulocytes) by manual Gentra Puregene. DNA was isolated from fibroblasts and buccal cells by phenol-chloroform extraction. TL was measured by Q-PCR of DNA from the three tissue types, and by flow-FISH in WBC subsets (lymphocytes and cell types matched to the types of WBC used in DNA extraction). We used the Wilcoxon signed-rank test to compare the median ranks of paired TL, and Spearman rank correlation coefficient to measure the strength of the associations between these measurements. ResultsTL in patients with DC was significantly (p<0.01) shorter than in patients with other IBMFS in all Q-PCR measurements of DNA from blood, fibroblasts, buccal cells; and flow-FISH WBC lymphocytes and matched cell types of WBC used in DNA extraction (86% were from granulocytes). Across all disorders, the median Q-PCR TL was longer in fibroblast and buccal cell DNA when compared with peripheral blood DNA (overall T/S ratio= 1.42 and 1.16 vs. 1.05, p=0.0002, 0.002, respectively). Although the absolute values varied, we observed in all IBMFS overall statistically significant (p≤0.001) intra-individual correlations in TL measured by Q-PCR in blood and fibroblast (r=0.67), blood and buccal cells (r=0.77), as well as fibroblast and buccal cells (r=0.67). When stratifying by disease subtype, statistically significant (p<0.05) correlations of Q-PCR in blood and buccal cells (r=0.9), and fibroblast and buccal cells (r=0.9) were observed in DC, and Q-PCR in blood and fibroblasts (r=1.0) in SDS. None of the Q-PCR tissue correlations reached statistical significance in FA or DBA. In addition, overall statistically significant (p≤0.01) correlations between TL in flow-FISH WBC subsets and Q-PCR in blood, fibroblast and buccal cells were observed. The correlations were driven mainly by the correlations with Q-PCR in blood and buccal cells in DC patients and with Q-PCR in fibroblasts in SDS patients. ConclusionsOverall Q-PCR TL was correlated between blood, buccal cells and fibroblasts and in comparison with flow-FISH TL, but there was some variability within different IBMFS. The poor correlation between tissues in FA and DBA might reflect blood-specific TL attrition in response to bone marrow stress. Most important, the correlation profile observed in DC suggests that the heritability of TL in DC is tissue independent. Blood or tissue Q-PCR TL appear to be useful in identifying individuals with DC who are unable to provide a fresh blood sample (e.g. epidemiologic studies or patients after bone marrow transplantation), although larger studies are needed to confirm or refute these findings. When possible, flow-FISH TL in subsets of leukocytes remains the modality of choice for the diagnosis of DC. DisclosuresLansdorp:Repeat Diagnostics Inc.: Equity Ownership.