Standard Flow Cytometry Research Articles

A New Method for Identifying Stem-Like Cells in Esophageal Cancer Cell Lines

Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, new techniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques.

Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer

Correct cell enumeration and differential analysis of body fluids are important in the diagnosis and management of several diseases. Currently, microscopic analysis is still considered the "gold standard". The aim of the present study was to evaluate the analytical performance of the CELL-DYN Sapphire hematology analyzer for automated differentiation of cells in serous fluids and to explore whether manual analysis of the raw data files could improve the differential count compared with reference microscopy. A total of 105 serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using standard whole-blood algorithm. Additionally, we performed optimized manual gating of the Sapphire raw data file using standard flow cytometry software. The standard Sapphire algorithm showed substantial deviations from the reference microscopic differentiation: polymorphonuclear cell counts were too high because they contained some monocytic cells. However, when optimized manual gating strategy is used, a good correlation and negligible bias were found. We have demonstrated that with a modified algorithm, CELL-DYN Sapphire will provide reliable identification and enumeration of blood cells in peritoneal and pleural fluids.

Fine Tuning of Surface CRLF2 Expression and Its Associated Signalling Profile in Childhood B Cell Precursor Acute Lymphoblastic Leukemia

Abstract 1409▪▪This icon denotes a clinically relevant abstractCytokine receptor-like factor 2 (CRLF2) alterations occur in 8% of unscreened children with B cell precursor acute lymphoblastic leukemia (BCP-ALL). The prognostic impact is still controversial and likely to be dependent on different clinical protocols. Biologic studies may facilitate the identification of factors contributing to these discrepant results. Moreover a better understanding of the biochemical mechanism(s) underlying such aberrancies may provide a more effective selection of patients that could benefit of signal transduction inhibitors treatments. In the present study we evaluated CRLF2 genomic rearrangements and the signalling profile involving pJAK, pSTAT5 and pS6 signalling pathways, known to be associated with CRLF2 rearrangements (Tasian SK et al, Blood 2012).A total of 60 (54 consecutive fresh and 6 thawed) diagnostic bone marrow samples from children with BCP-ALL enrolled in AIEOP-BFM-ALL trials were analyzed for CRLF2 surface expression by flow cytometry (FCM). CRLF2 transcript levels as well as CRLF2 aberrations (i.e. P2RY8-CRLF2) were analyzed by molecular methods as recently described (Palmi C et al, Leukemia 2012). TSLP-induced (10 ng/mL for 30 minutes) pSTAT5 and pS6 response were evaluated in CD7-/CD19+/CD10+/CD45low blasts by phosphoflow cytometry in thawed mononuclear cells obtained after ficoll gradient centrifugation.Eight out of 60 (13.3%) samples were FCM CRLF2 ’positive’ (mean value of CRLF2+ cells = 87.5%) with a bright or intermediate fluorescence distribution. However, further cases showed peculiar CRLF2 expression: 2/60 (2.25%) were ’partially positive’ (mean CRLF2+ cells = 2.25%), with two clearly distinguished blast populations (one negative and one positive); 9/60 (15.00%) were ’dim positive’ (mean CRLF2+ cells = 3.24%) showing a clear shift-to-right distribution compared to negative control (normal B lymphocytes CD7-/CD19+/CD10-/CD45++), and 41/60 (68.30%) were fully ’negative’ (CRLF2+ cells <1%). Parallel RT-PCR analysis of CRLF2 expression was performed in 51 samples: all FCM-positive samples were over expressed by PCR (i.e. fold change of >20 compared to the median value of almost 500 samples tested) whilst CRLF2 ’dim positive’ or ’partially positive’ had a PCR fold change of < 20. Interestingly, 29/32 CRLF2 ’negative’ were concordantly negative by PCR, however, in 3/32 samples a fold change of >20 was obtained. Of note, in these 3 discordant cases P2RY8-CRLF2 was detected as a very weak PCR amplification (n=2) or PCR negative (n=1), suggesting the presence of minor rearranged cell populations.Aberrant pSTAT5 response was observed in all CRLF2 ‘positive' analyzed samples (n = 8) compared with CRLF2 ’negative’ cases (n = 9) showing 59.7% and 5.4% of pSTAT5+ cells (means), respectively (p =0.00001). Interestingly CRLF2 ’dim positive’ cases (n = 5) had an intermediate level of pSTAT5 response (mean 18.1%) significantly different compared to both CRLF2 ’positive’ and CRLF2 ’negative’ cases (p <0.05). Our results are concordant with recent findings by Taisan et al, although with some differences. We found that IL-7 (100 ng/mL) induced p-STAT5 response at variable levels in BCP-ALL blasts regardless of CRLF2 expression. Also TSLP-induced pS6 response, in addition to pSTAT5, was aberrant in CRLF2 ’positive’ cases compared to CRLF2 ’negative’ ones (mean 26.9 vs. 0.2, p = 0.0001), yet CRLF2 ’dim positive’ cases showed intermediate mean level of pS6 response being 8.3%. All CRLF2 ’dim positive’ cases were negative (or undetectable) for P2RY8-CRLF2 deletion.In conclusion, we confirm and further extend that TSLP mediated signal transduction induces aberrant JAK-STAT and PI3K-mTOR signaling pathways in CRFL2 mutated or over expressed BCP-ALL cases. Of note, cases with dim positivity of CRLF2 (usually considered negative by standard flow cytometry criteria) and negativity for P2RY8-CRLF2 deletion showed an active pSTAT5 and pS6 signaling at intermediate level between CRLF2 'positive' and CRLF2 'negative'. Whether it refers to the pattern of minor clones or to the presence of additional mechanism(s) driving aberrant signal transduction, are still under investigation. Overall, these findings may be relevant for diagnosis and prognosis in CLRF2 positive BCP-ALLs; they further support that signal transduction inhibitors may have therapeutic relevance in this setting. Disclosures:No relevant conflicts of interest to declare.

10 Regulation of Monocyte-Endothelial Cell Interactions by Neutrophil-Derived Microparticles

Rationale Monocytes play a major role in atherosclerosis progression through migration into the arterial wall. Neutrophil involvement in atherosclerosis was previously only thought to be via enzymatic weakening of the fibrous cap. However, neutrophil depletion can delay atherogenesis and conversely increasing circulating neutrophil enhance plaque progression in mice. Lack of evidence for the presence of neutrophils in atherosclerotic plaques makes their role in disease progression less clear. We have found neutrophil-derived microparticles increase migration of neutrophils. Our hypothesis: neutrophil-derived microparticles increase endothelial cell-monocyte interactions and facilitate monocyte transendothelial migration. Methodology Neutrophils were incubated with various agents to stimulate microparticle formation. Microparticles were characterised and quantified using a novel, standardised flow cytometry method and electron microscopy. Monocytes were migrated to MCP-1 across CAEC incubated with neutrophil-derived microparticles using a transendothelial migration assay. Cytokine release and adhesion molecule expression by hCAEC was investigated using a cytometric bead array and fluorescent antibody binding respectively. Microparticle adhesion to hCAECs was investigated under static and flow conditions. Findings Neutrophil-derived microparticles were between 0.5 and 0.9µm. The most abundant surface markers were Annexin V with 20-30% positive microparticles, CD66c (10–15%) and CD18 (5–15%). Low levels of CD11b, CXCR2, and L-selectin were observed. More microparticles were produced in response to fMLP and aLDL compared to PBS. Microparticles bind to and induce MCP-1 release from hCAEC. Monocyte migration increased upon hCAEC incubation with neutrophil-derived microparticles and the extent of the increase was dependent on the stimulus used with aLDL>fMLP>PBS. This highlights a role for microparticles in endothelial activation.

A novel image-based cytometry method for autophagy detection in living cells

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID® Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities.

Evaluation of the dual IFNγ/IL-2 fluorospot-assay with flow cytometry for detection of HLA-restricted HIV-specific T-cell responses in HIV controllers

Background: The IFNγ ELISpot assay is used widely for high through-put screening of HIV-specific responses in studies of HIV infection and vaccine studies. However, dual production of IFN/IL-2 and increased proliferative capacity may be associated with better natural control of HIV infection. We evaluated a novel fluorospot assay enabling the identification of dual IFNγ/ IL-2 producing antigen-specific cells and compared it with intracellular cytokine staining by standard flow cytometry in individuals with natural control of HIV-infection. Methods: PBMC from five untreated HIV-infected individuals were stimulated overnight with HIV peptides or controls in dual IFN/IL-2 pre-coated plates. Peptide arrays were specifically selected based on the individual HLA type. Secreted IFNγ and IL-2 were detected using fluorescent-conjugated antibodies. Fluorescent spots were enumerated on the iSpot AID reader. Responses were considered positive if >50 spots/million cells SFU after background subtraction.Positive responses were then evaluated by flow cytometry using the Gallios flow cytometer. Results: Dual IL-2/IFNγ producing cells were detected to anti-CD3- stimulated PBMC from all patients. IFNγ responses alone were detected to 35 of 136 HLA-restricted peptides tested (median =73, 52-4190 SFU) across the 5 patients (1/19, 5/19, 7/28, 9/15 and 13/55 for each patient), while IL-2 responses were either low grade or undetectable for the majority of HIV peptides tested. Dual IFNγ/IL-2 producing HIV-specific T cells were not detected using the fluorospot assay. 24/35 peptides induced CD8 T cell- IFNγ production by flow cytometry. Conclusion: HIV-specific mono-IFNγ responses were detected using the novel fluorospot assay. However limited HIV-specific dual IFNγ/IL-2 responses were detected in this patient group. A greater number of epitope-specific positive responses were detected in the fluorospot compared with flow cytometry suggesting the fluorospot may be more sensitive in detecting a greater breadth of epitope-specific T cell responses, and therefore better for screening purposes than flow cytometric methods.

Predicting outcome based on minimal residual disease (MRD) using multiparameter flow cytometry (FC) in acute myeloid leukemia (AML) patients (Pts).

6565 Background: Most AML pts will experience relapse caused by persistent leukemic cells during complete remission (CR). The aim of our study was to predict outcome in AML pts in CR by assessing their MRD using standard 4-colour FC panels available at our institute. Methods: We queried our AML database between 1/2004 to 10/2010 for newly diagnosed untreated AML pts &gt;18 years of age who achieved CR after one induction regimen. Treatment included 7+3 or similar intensive approaches. The gating strategy used a series of Boolean regions that best defined the diagnostic abnormal population based on its expression patterns using three 4-colour FC panels (F7-CD38,CD10,CD19,CD34; F9-C11b,CD33,CD13,CD34; F38-CD15,CD56,CD7,CD34). The same regions were applied to post-induction samples, and the number of residual events was determined. Results: 140 AML patients with a median age of 64 (range 24-93) years, including 74 females (52.8%) were analyzed; 67 (47.8%) patients relapsed. Eighty-eight (62.8%) pts were CD34-positive (CD34+) at diagnosis. Normal karyotype was detected in 27 (30.7%): FLT-3 ITD was detected in 4/23 (17.4%) and NPM-1 mutation was detected in 1/15 (0.07%) samples. Median follow-up for CD34+ pts was 17.9 months. Forty-two (30%) pts underwent stem cell transplantation (SCT) in first CR: 31 (22.1 %) were allogeneic and 11 (7.9%) autologous. In multivariate analysis, a detectable MRD in any of the 3 panels at or beyond week 16 among CD34+ AML pts in CR was significantly associated with inferior relapse free survival (F7: P=0.035 , F9: P=0.027, F38: P=0.042). In addition, MRD ≥ week 16 using panel F9 was also significantly associated with inferior overall survival (P=0.0224). In pair-wise comparison, those who were MRD-negative ≥ week 16 did not benefit from SCT compared to the non-SCT group. Similar analyses among CD34– AML pts did not achieve statistical significance. Conclusions: MRD+ by FC in CD34+ AML is an independent prognostic factor and can identify pts who may benefit from SCT/more intensive therapy to maintain remission. However, the value of studying MRD in CD34– AML requires further consideration. More effort is needed to identify stem cells in CD34– AML.

Modeling SDF-1–induced mobilization in leukemia cell lines

The stromal cell-derived factor 1 (SDF-1) is essential for circulation, homing, and retention of hematopoietic stem cells in the bone marrow. Present evidence indicates that this factor might play an important role in leukemia cells as well. The aim of this study is to present a model of SDF-1-induced mobilization using leukemia cell lines. CXCR4 expression was compared in Kasumi-1, Jurkat, HL-60, KG-1a, and K562 cells by flow cytometry and Western blot. Migration was analyzed with Transwell assays, and adhesive cell-cell interaction was quantified with a standardized adhesion assay and flow cytometry. CXCR4 was expressed by all leukemic cell lines analyzed, although surface expression of this receptor was found in Kasumi-1 and Jurkat cells only. Correspondingly, SDF-1α effects on migration and cell-cell adhesion were observed in Kasumi-1 and Jurkat cells only, and this could be blocked by AMD3100 in a reversible manner. We have provided evidence that SDF-1α acts as a chemotactic and chemokinetic agent. In addition, surface expression of integrin-β2, activated leukocyte cell adhesion molecule and N-cadherin decreased after stimulation with SDF-1α. SDF-1α affects cell-cell adhesion and migration only in leukemia cells on which the CXCR4 receptor is present on the surface. An SDF-1 gradient is not necessarily required to induce migration, as chemokinesis can also occur. Upon stimulation with SDF-1, CXCR4 promotes modifications on the surface pattern of adhesion molecules, which have an influence on adhesion and migration.

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Abstract Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with &amp;gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p&amp;lt;0.05) and Raji vs Raji-4RH (p&amp;lt;0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 516. doi:1538-7445.AM2012-516

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high‐performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage

All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high-performance liquid chromatography (HPLC). RhD-negative women who deliver RhD-positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF-positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti-D FC was performed to quantitate fetal D-positive cells in D-negative women and compared with FMH estimated by AE and HbF FC. AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti-D FC are discrepant or when there is maternal and fetal RhD compatibility.

Impact of methylprednisolone in priming solution of cardiopulmonary bypass on anti-inflammatory CD163 receptor during cardiac surgery

We evaluated the influence of methylprednisolone in cardiopulmonary bypass fluid on scavenger receptor for hemoglobin CD163 molecule expression on monocytes of patients who underwent elective coronary artery bypass grafting with cardiopulmonary bypass with either exposure to methylprednisolone present in the cardiopulmonary bypass fluid (20 patients), or without methylprednisolone in the cardiopulmonary bypass fluid (22 patients) and operated on without cardiopulmonary bypass (42 patients). The dynamics of CD163 expression was also followed in patients operated on without cardiopulmonary bypass. This study was a retrospective analysis of a comparison of two studies. The expression of CD163 was determined quantitatively by standardized flow cytometry technique. The similarities in the dynamics of CD163 monocyte expression, comparing the patients operated on with or without cardiopulmonary bypass, were found. Compared to the preoperative level, CD163 monocyte expression was significantly elevated on the 1(st) postoperative day. Monocyte CD163 expression on the 1(st) postoperative day was evidently similar in both groups of patients operated without cardiopulmonary bypass (median value of mean fluorescence intensity (MFI) 18,896; interquartile range from 27,538 to 57,711; median value of MFI 18,863; interquartile range from 16,514 to 26,559; n.s.), suggesting high reproducibility of our flow cytometric method; the monocyte CD163 expression was significantly higher (median value of MFI 37,902; interquartile range from 27,538 to 57,711) on the 1(st) postoperative day in patients exposed to methylprednisolone compared to patients without this exposure (median value of MFI 20,995; interquartile range from 16,321 to 29,623) (p<0.001). We concluded that the expression of hemoglobin scavenger receptor CD163 on monocytes of cardiac surgical patients is induced by methylprednisolone present in cardiopulmonary bypass fluid.

A new AQP1 null allele identified in a Gypsy woman who developed an anti‐CO3 during her first pregnancy

The Colton blood group antigens are carried by the AQP1 water channel. AQP1(-/-) individuals, also known as Colton-null since they express no Colton antigens, do not suffer any apparent clinical consequence but may develop a clinically significant alloantibody (anti-CO3) induced by transfusion or pregnancy. Identification and transfusion support of Colton-null patients are highly challenging, not only due to the extreme rarity of this phenotype, the lack of appropriate reagents in most laboratories, as well as the possibility of confusing it with the recently described CO:-1,-2,3,-4 phenotype where AQP1 is present. This study investigated a new Colton-null case and evaluated three commercially available anti-AQP1s to identify Colton-null red blood cell samples. The Colton-null phenotype was investigated by standard serological techniques, AQP1 sequencing, immunoblot and flow cytometry analyses. We identified and characterized the Colton-null phenotype in a Gypsy woman who developed an anti-CO3 during her first pregnancy. After developing a simple and robust method to sequence AQP1, we showed that she was apparently homozygous for a new AQP1 null allele, AQP1 601delG, whose product is not expressed in her red blood cells. We also established the Colton specificity of three commercially available anti-AQP1s in immunoblot and/or flow cytometry analyses. This Gypsy woman represents the sixth Colton-null case characterized at the serological, genetic and biochemical levels. The validation here of new reagents and methods should facilitate the identification of Colton-null individuals.

High-Sensitivity Flow Cytometry Provides Access to Standardized Measurement of Small-Size Microparticles—Brief Report

Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels. Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 μm) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8- to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information. Recent improvements in FCM provide access to previously undetectable MP and represent a new opportunity to enhance their impact as biomarkers in clinical practice.

Imaging flow cytometry: a subtle and depth analysis of molecular mechanisms

The ImageStreamX is an innovative instrument that takes advantage of imaging flow cytometry, a novel technique that combines the speed, statistical power, and fluorescence sensitivity of flow cytometry with the functional insights of high resolution microscopy to give the most insightful cell analysis possible [1]. Among the wide range of applications, in our laboratory we study the human gingival fibroblasts (HGF) response to resin-based materials commonly used in dentistry, in terms of membrane molecule expression, intracellular signal transduction and cell death and apoptosis. Our experimental model is thought to resemble the oral cavity by cultivating the cells in the presence of saliva flow and microrganisms commonly present in vivo. As regards surface antigens expression, IDEAS image analysis software allows to virtually quantitate anything you can see using the software package’s numerous predefined fluorescence and morphologic parameters. Regarding the signal transduction, the IDEAS software package quantifies nuclear translocation events by automatically correlating the images of the transcription factor and the nucleus using the Similarity score. As of cell death and expression, Image StreamX can perform any standard flow cytometry assay, i.e. Annexin-V/PI one, with the added value of visual confirmation.

Infection of bone marrow cells by dengue virus in vivo

Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41(+)CD61(+) cells, regardless of CD34 and CD45 expression. The CD61(+) cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting-assisted isolation of CD61(+) cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.

Long-Term Detection of Fluorescently Labeled Human Mesenchymal Stem CellIn VitroandIn Vivoby Semi-Automated Microscopy

The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.

The Uptake of Recombinant Glucocerebrosidases by Blood Monocytes From Type 1 Gaucher Disease Patients Is Heterogeneous

Abstract 1102 Introduction:Gaucher disease (GD) is due to an inborn deficiency of glucocerebrosidase (GC), that leads to the accumulation of glucosylceramide in monocytes/macrophages, known as “Gaucher cells”, which are thought to be responsible for a wide range of symptoms. Imiglucerase (CEREZYME®, Genzyme Corporation) (IMI) is the first line treatment of type 1 GD patients. Two new biosimilar agents, velaglucerase-alfa (VPRIV®, Shire Human Genetic Therapies, Dublin, Ireland) (VEL) and taliglucerase-alfa (PROTALIX®, Biotherapeutics, Pfizer) (TAL), have been described as being similar to IMI but differing slightly in glycan structure which could have an impact on macrophage uptake and thus on therapeutic efficacy. However, the ability of native GD cells to capture recombinant enzymes (REs) remains unknown. We used blood monocytes (Mo) from GD patients and healthy donors (HD) as a model to compare the uptake of the three REs. Patients and methods:After informed consent was obtained, cells were obtained from healthy donor blood (n=34). The left over part of biological samples collected for routine analysis from Gaucher patients (n=6) could be used for research because patients had been informed and did not verbally express any disagreement. Mononuclear cells were incubated with three concentrations of each RE (0.1, 0.5 and 1 U/ml) at 37°C for 30 minutes and one hour and washed twice with PBS to eliminate exogenous RE before evaluation of intra-cellular GC activity by standardized flow cytometry as previously described (Berger J. et al., Br J Haematol 2010) Results:Firstly, we confirmed that GD Mo (n=6) showed a marked enzyme deficiency (about 7% of the normal endogenous activity) as compared to normal glucocerebrosidase activity (GCA) (n=34) Then we observed a dose-dependent in vitro uptake of IMI, TAL and VEL in Mo from 4 untreated GD patients. However, the intra-monocyte (IMo) GCA of TAL was systematically lower than that of IMI and VEL.Case analysis showed an inter-patient heterogeneity, with the highest increase of intra-monocyte enzyme activity for all REs in patient #2 and the lowest in patient #3; this observation was confirmed in vivo by analysis of Mo 15 min. after the end of the first IMI infusion (× 38 and × 9 endogeneous IMoGCA respectively) whereas patient #2 had received a lower dose of enzyme (45 U/kg/2 weeks vs 60 U/kg/2 weeks). Interestingly, patient #2 showed mild GD suffering only thrombocytopenia and asthenia while patient #3 had an aggressive form of GD, with bone disorders (aseptic osteonecrosis, bone infarction and pseudarthrosis after traumatic fracture). Patient #2 responded rapidly, with improved thrombocytopenia (68%) and increased hemoglobin level (+1.9g/dL) from M3 but hematological response for patient #3 could not be evaluated because initial parameters had been normal in this patient who had been splenectomized 20 years ago. Because of the chitotriosidase deficiency of patient #2, we used plasma CCL18 as a biomarker; its kinetic of decrease was clearly more rapid than for patient #3. Similarly, correction of glycosylated-ferritin was better than in patient #2. The two other patients with IMoGCA values close to that of patient #2 had non-progressive disease not requiring treatment (patient #1), or had thrombocytopenia that improved over the expected period of time (patient #4). Discussion:In conclusion, this study shows inter-patient variability in the ability of blood Mo to store recombinant enzymes which to our knowledge has not been previously reported. This was confirmed in vivo 15 min. after the start of the infusion. This variability could partially explain the heterogeneity of GD response to enzyme replacement therapy. In this small series, the least aggressive disease corresponded to the highest intra-monocyte GCA and the most aggressive disease to the least intra-monocyte GCA. Six-month follow-up showed differences in change in biological biomarkers suggesting a relationship between intra-monocyte GCA and disease response. Furthermore in this in vitro GD Mo model, all the compounds available are not similar, even if their chemical structures are only slightly different with no relationship with the expression of the CD206 (data not shown). Another RE influx mechanism could exist and influence enzyme replacement therapy. These findings could help in customizing replacement therapy. Disclosures:Belmatoug:Genzyme: Consultancy; Shire: Research Funding. Berger:Shire: Consultancy; Genzyme: Consultancy, Research Funding.

Graft-Versus-Lymphoma Effect After Non-Myeloablative Allogeneic Transplant Induces Molecular Remission Assessed by High-Throughput Sequencing of T Cell Receptor in Patients with Advanced Stage Mycosis Fungoides and Sezary Syndrome

Abstract 3114Mycosis fungoides (MF) is a mature T-cell lymphoma arising primarily from the skin. Patients with advanced stage disease either with skin tumor lesions or its leukemic form, Sezary syndrome (SS), consistently require therapy for disease control and symptom relief. Currently, there is no curative therapy and none of the available therapies provides a long-term remission. We have performed non-myeloablative allogeneic transplant in 11 MF/SS patients using total skin electron beam therapy (TSEBT), total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) preparative regimen in an attempt to provide prolonged disease control. The median age was 63 years (range 20–73). All but one patient had stage IV disease at the time of preparative regimen. The median number of prior systemic therapies was 5 (range 2–8). Eight patients achieved complete response after transplant and 3 patients had partial response (ORR 100%). However, 4 patients experienced disease progression post transplant, requiring additional therapy. Patients tolerated the transplant extremely well. Only one patient developed grade 2 acute GVHD (skin), and another patient developed extensive chronic GVHD (skin, oral, GI). All patients were alive at the last evaluation (median follow-up, 10.6 months).The current method for monitoring residual disease in MF/SS uses flow cytometry to detect circulating Sezary cells and pathological evaluation of skin biopsy samples. However, there are no MF/SS specific tumor markers that can easily and consistently differentiate malignant clones from normal T cells and these standard diagnostic tools lack sensitivity in detecting minimal residual disease (MRD). Here, we tested whether high-throughput sequencing of T cell receptor (TCR) provides a new tool for monitoring MRD after allogeneic transplant. Genomic DNA was extracted from either peripheral blood mononuclear cells or selected skin biopsy samples. The rearranged VDJ of TCR ß was amplified using V ß-specific forward and J ß-specific reverse primers. The Illumina GA2 system generated up to 1, 000, 000 reads of 54 base pairs, covering the entire CDR3 lengths. Raw sequence data were processed to remove PCR/sequencing errors, and a nearest neighbor algorithm was used to collapse the data into unique sequences (Blood 2009, 114 :4099). Of blood samples from six SS patients, malignant clone was identified in all 6 cases by a dominant unique TCR ß CDR3 sequence. At the time of preparative regimen, 3 patients had measurable circulating Sezary cells by standard flow cytometry and pathological evaluation of peripheral blood smear. TCR ß sequencing data showed that malignant clones contributed to 69%, 81% and 68% of circulating T cells in these 3 patients, respectively. The other 3 patients did not have detectable circulating Sezary cells by standard diagnostic tools. However, 8.47%, 0.38% and 0.22% of the TCR ß sequences were from the malignant clones in these 3 cases at the time of preparative regimen. The percentage of malignant T cell clones decreased in all cases immediately after transplant. Three patients eventually achieved molecular remission. Two of them cleared the malignant clone at day+30 and the third cleared the MRD at day+270. The follow-up is still short to determine whether achieving molecular remission correlate with better clinical outcome. We have also started to perform TCR ß sequencing of skin biopsy samples to assess the MRD status in the skin of MF patients. In addition to providing highly sensitive and specific MRD quantification, deep sequencing of TCR also revealed vital information on the T cell repertoire reconstitution after allogeneic transplant. Based on our preliminary data, adaptive transfer of donor TCR repertoire clearly occurred immediately after allogeneic transplant and the TCR diversification slowly appeared months later.In summary, we have shown a safe and effective unique preparatory regimen for non-myeloablative allogeneic transplant in patients with advanced stage MF and SS. We also demonstrated the utility of high throughput sequencing of CDR3 of TCR ß in assessing MRD status after therapy with utmost sensitivity and specificity, and for the first time, showed a therapy resulting in molecular remission in this patient population. Disclosures:No relevant conflicts of interest to declare.

High-Sensitivity Flow Cytometry Testing for Paroxysmal Nocturnal Hemoglobinuria in Children with Cytopenia: A Single Center Study

Abstract 2398 Background:Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by expansion of cells with complete or partial loss of glycosyl phosphatidyl-inositol-anchored proteins. PNH usually presents with one or more of three clinical manifestations: intravascular hemolysis, thrombosis, or acquired bone marrow failure [aplastic anemia (AA) or myelodysplastic syndrome (MDS)]. Flow cytometry has become the gold standard for the diagnosis of PNH, particularly with the recent publication of guidelines for the diagnosis and monitoring of PNH and related disorders in 2010. PNH occurs rarely in children, and, consequently, the published literature regarding PNH in this pediatric population consists only of small case series, making it difficult to extrapolate the frequency of which PNH clones are identified. Moreover, no studies are available on the incidence of PNH clones in children with MDS and acquired aplastic anemia (AAA). We, therefore, sought to determine how frequently a high sensitivity FLAER-based assay, with a sensitivity of 0.01%, would detect PNH clones in children with cytopenias. Method and Results:The study period was from December 2010 to July 2011. PNH testing was performed using a high sensitivity FLAER based assay according to published guidelines using the combination of FLAER/CD64/CD15/CD33/CD24/CD14/CD45 for WBC testing and CD235a/CD59 for RBC testing. There were 31 peripheral blood samples from 29 patients (17 males/12 females) ranging in age from 4 months to 17 years (median, 10 years). All patients were tested for PNH because of cytopenia [pancytopenia (n = 14) and uni- or bicytopenia (n = 15)]. Patients had a mean Hgb of 10.7 gm/dL, mean ANC of 2.66 X103/uL and mean platelet of 115 X103/uL. Review of medical charts revealed the following clinical diagnoses: classic PNH - episodic hemolytic anemia with persistent thrombocytopenia (1), severe AA (SAA, 8), SAA with myelofibrosis (1), MDS (1), Fanconi anemia (1), chronic thrombocytopenia (2), refractory iron deficiency anemia (1), bone marrow suppression likely due to virus/medication (1), parvovirus infection (1), Copper deficiency (1), systemic lupus erythematosus (SLE, 1), and cytopenia of unknown etiology (10). Of note, all patients with AAA had SAA. PNH clones were identified in 6 out of 29 patients (20%): minor clones (<1% PNH population) in 3 patients: average clone sizes 0.12% [range 0.02–0.25] granulocytes (G), 0.51% [0.20–0.99] monocytes (M), and 0.08% [0.04–0.14] red blood cells (RBCs), and major clones (>1% PNH population) in 3 patients: average clone sizes 31.11% [3.98–67.58] G, 31.98% [6.15–71.1] M, and 14.76% [1.19–38.03] RBC, respectively, with ages ranging from 4 to 17 years. Patients who were identified to have minor PNH clones all presented with pancytopenia. Two were diagnosed with SAA; the cause of pancytopenia in the third patient is currently under investigation. None of patients with minor PNH clones had evidence of hemolysis or thrombosis. The three patients with major PNH clones had the following: Classic PNH with hemolytic anemia (1), SAA with PNH clones detected at the time of SAA diagnosis (1), and SAA with PNH clones detected 20 months after immunosuppressive therapy (1). The latter two patients did not have evidence of hemolysis or thrombosis. Of the 10 patients with a diagnosis of SAA or MDS, PNH clones were identified in 4 (40%) patients (2 with minor clones, 2 with major clones). Conclusions:This is the first study to describe the utility of using a standardized high-sensitivity FLAER-based flow cytometry assay to identify PNH clones in children. This is also the first study describing the prevalence of PNH clones in children with MDS and AAA. The identification of a PNH population in 40% of the MDS and AAA cases emphasizes the need for PNH testing in all children with these disorders using a high-sensitivity FLAER based flow cytometry assay. A low sensitivity assay would have missed 2 patients with minor PNH clones. This finding may be of significance considering SAA or MDS patients with PNH clones are more likely to respond to immunosuppressive therapy. Further studies are needed to investigate the prevalence of PNH clones in this setting and its impact on disease manifestations, course, and outcomes in children. Disclosures:No relevant conflicts of interest to declare.