Cytometry A. 2010 Sep 2;
Lin CC, Huang WL, Su WP, Chen HH, Lai WW, Yan JJ, Su WC

Single cell phospho-specific flow cytometry (SCPFC) enables the investigation of signaling network interactions and the categorization of disease outcome. While this method has been successfully used to study hematologic disorders, its application on solid tumors has not been examined. This study aimed to demonstrate the ability of SCPFC to detect dynamic changes of Tyrosine phospho-Stat1 (pStat1) in solid tumor models and in human tumor samples. In the human lung cancer cell line PC14PE6/AS2, the fluorescence intensity changes of pStat1 after IFN-gamma stimulation were compatible to results obtained by Western blot analysis. In metastatic animal models, cancer cells from subcutaneous tumors, malignant ascites, and peritoneal tumors responded to IFN-gamma. The pStat1 was activated in these cells after IFN-gamma stimulation, with a 1.5- to 2.5-fold increase in fluorescence intensity compared to the unstimulated control. To examine the potential clinical application of SCPFC, cancer cells were collected from malignant pleural effusions (MPEs) of lung cancer patients to observe the activation of pStat1 after IFN-gamma stimulation. Cell apoptosis after cisplatin treatment was evaluated by Annexin V staining, which showed that MPE cancer cells with higher pStat1 changes after IFN-gamma stimulation were more resistant to cisplatin. In conclusion, there is a preliminary application of SCPFC to solid tumors and links to drug sensitivity are promising. (c) 2010 International Society for Advancement of Cytometry.

Gut. 2010 Aug 30;
van Wanrooij RL, Schreurs MW, Bouma G, von Blomberg BM, Tack GJ, Verbeek WH, Mulder CJ

Cytometry A. 2010 Sep; 77(9): 831-9
Estes ML, Mund JA, Mead LE, Prater DN, Cai S, Wang H, Pollok KE, Murphy MP, An CS, Srour EF, Ingram DA, Case J

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133(+) subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133(+) proangiogenic CHSPCs to AC133(-) nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature. (c) 2010 International Society for Advancement of Cytometry.

Fertil Steril. 2010 Aug 24;
Niu ZH, Huang XF, Jia XF, Zheng J, Yuan Y, Shi TY, Diao H, Yu HG, Sun F, Zhang HQ, Shi HJ, Feng Y

Spermatozoa viability tests based on dual-color flow cytometry after staining with Sybr-14/propidium iodide were performed on 44 men with complete asthenospermia for primary ciliary dyskinesia (PCD) screening, and seven were identified with PCD by electron microscopy of ultrastructural ciliary defects. Six PCD patients underwent eight intracytoplasmic sperm injection therapy cycles using ejaculated sperm or testicular sperm, obtaining a mean fertilization rate of 46.6%, with three healthy babies born and one in utero at the time of writing.

J Clin Microbiol. 2010 Aug 25;
Pieretti B, Brunati P, Pini B, Colzani C, Congedo P, Rocchi M, Terramocci R

Urinary Tract Infection (UTI) is a widespread disease and thus, the most common samples tested in diagnostic microbiology laboratories are urine samples. The "gold standard" for diagnosis is still bacterial culture but a large proportion of samples are negative. Unnecessary culture can be reduced by an effective screening test. We have evaluated the performances of a new urine cytometer, the Sysmex UF-1000i (Dasit), in 703 urine samples submitted to our laboratory for culture. We compared bacteria and leukocytes (WBC) count performed with the Sysmex UF-1000i against colony-forming units per millilitre (CFUs/mL) quantification on CPS agar to assess the best cutoff values. Different cutoff values of bacteria/mL and WBC/mL were compared to give the best discrimination. On the basis of results obtained in this study, we suggest, by using the Sysmex UF-1000i analyser as a screening test for UTI, that the cutoff values should be 65 bacteria/mL and 100 WBC/mL. Diagnostic performances in terms of Sensitivity (98.2%), Specificity (62.1%), Negative Predictive Value (98.7%), Positive Predictive Value (53.7%), Diagnostic Accuracy (73.3%) were satisfactory. Screening with the Sysmex UF-1000i is acceptable for routine use. In our laboratory we have reduced by 43% the bacterial culture and speeded up their reporting and decreased the inappropriate use of antibiotics.

Cytometry A. 2010 Sep; 77(9): 813

Cytopathology. 2010 Aug 23;
Schmid S, Tinguely M, Cione P, Moch H, Bode B

S. Schmid, M. Tinguely, P. Cione, H. Moch and B. Bode Flow cytometry as an accurate tool to complement fine needle aspiration cytology in the diagnosis of low grade malignant lymphomas Objective: Diagnosis of low grade non-Hodgkin B-cell lymphomas on cytological material may be problematic and in the past frequently required lymph node excision. We analysed our experience of the value of flow cytometry (FC) as an additional tool for the diagnosis of lymphoproliferative processes in the setting of a university cytology division with a busy fine needle cytology service. Methods: Consecutive cytological specimens with FC over a period of 3 years were retrospectively analysed and correlated with histology and follow-up if available. FC was performed with the following antibodies: CD3, CD4, CD8, CD2, CD7, CD19, CD5, CD10, CD23, lambda and kappa chains. Results: Of 299 probes (273 fine needle aspirations and 26 fluids from 285 patients), 179 cases (60%) were diagnosed as reactive, 91 cases (30%) as malignant or suspicious and 29 cases (10%) as inconclusive. The results of histological examination of the lymph nodes were available in 41 of 91 (45%) malignant or suspicious cases and in 13 of 179 (7%) reactive cytological diagnoses. Cytologically diagnosed malignancy was confirmed in all histologically examined cases. In 12 of 13 reactive cytological cases (92%), a benign process was diagnosed histologically. In 34 of 299 cases (11%) additional molecular investigations of B-cell clonality or specific translocations were performed. The lymphomas most frequently diagnosed were follicular lymphoma and lymphocytic lymphoma, followed by mantle cell and marginal zone lymphomas. Correlation with histology showed a sensitivity of 98% and a specificity of 100% for cytology in our series. Conclusions: FC is an important additional tool in the cytological diagnosis of lymphoproliferative disorders. The combined approach has a high diagnostic value that allows a reliable subclassification of low grade B-cell non-Hodgkin lymphomas.

Angiogenesis. 2010 Aug 24;
Sottnik JL, Guth AM, Mitchell LA, Dow SW

The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31(+) endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans.

Theriogenology. 2010 Aug 20;
Carvalho JO, Sartori R, Machado GM, Mourão GB, Dode MA

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means +/- standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 +/- 3.0, 58.2 +/- 3.0, and 60.9 +/- 3.3) than SX (29.6 +/- 1.3, 36.0 +/- 2.9, and 37.1 +/- 3.3), and SY (26.2 +/- 2.1, 36.4 +/- 2.9, and 37.5 +/- 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 +/- 3.2, 18.1 +/- 3.3, and 14.8 +/- 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.

J Immunol Methods. 2010 Aug 19;
Chen M, Chang JS, Nason M, Rangel D, Gall JG, Graham BS, Ledgerwood JE

Respiratory syncytial virus (RSV) is an important cause of respiratory infection in people of all ages, and is the leading cause of hospitalization in infants. Although commercially available monoclonal antibody is available for passive prophylaxis of neonates at risk of severe disease, there is no available vaccine to prevent RSV. Measurement of neutralizing activity will be a key endpoint for vaccine evaluation. Assessment of neutralizing antibody against RSV has been limited to traditional plaque reduction, which is time-consuming and inherently operator dependent and highly variable. Here, we describe a flow cytometry-based RSV-specific neutralization assay which is more rapid than traditional methods, highly sensitive and highly reproducible.

Eur J Haematol. 2010 Aug 19;
Schroers R, Baraniskin A, Heute C, Vorgerd M, Brunn A, Kuhnhenn J, Kowoll A, Alekseyev A, Schmiegel W, Schlegel U, Deckert M, Pels H

Abstract Reliable detection of leptomeningeal disease has the potential of facilitating the diagnosis of CNS lymphoma and is important for therapeutic considerations. Currently, the standard diagnostic procedure for detection of lymphoma in the cerebrospinal fluid is cytopathology. In order to improve the limited specificity and sensitivity of cytopathology, flow cytometry has been suggested as an alternative. Here, we evaluated multi-parameter flow cytometry in combination with conventional cytopathology in CSF samples from 30 patients with primary and 7 patients with secondary CNS lymphoma, respectively. Overall, in 11 of 37 (29.7%) CNS patients, lymphoma cells were detected in CSF by flow cytometry, while cytopathology was less sensitive displaying unequivocally malignant CSF cells in only seven of all 37 (18.9%) patients. Six (16.2%) patients showed cytopathological results suspicious of lymphoma; however, in only one of these patients the diagnosis of CSF lymphoma cells could be confirmed by flow cytometry. In primary CNS lymphomas (PCNSL), seven of 30 (23.3%) patients were positive for CSF lymphoma cells in flow cytometry, in contrast to four (13.3%) PCNSL patients with definitely positive cytopathology. In summary, our results suggest that multi-parameter flow cytometry increases the sensitivity and specificity of leptomeningeal disease detection in CNS lymphomas. Both methods should be applied concurrently for complementary diagnostic assessment in CNS lymphoma patients.

Cytometry A. 2010 Aug 18;
Reynolds MR, Piaskowski SM, Weisgrau KL, Weiler AM, Friedrich TC, Rakasz EG

Deciphering the complex interactions between human and simian immunodeficiency viruses (HIV/SIV) and their host cells is crucial to the development of improved therapies and vaccines. Investigating these relationships has been complicated by the inability to directly analyze infected cells among freshly isolated peripheral blood lymphocytes. Here, we describe a method to detect cells productively infected with SIVmac239 ex vivo from the blood or lymph nodes by flow cytometry. Using this method, we show a close correlation between the frequency of productively infected cells in both sample type and the plasma viral load. We define that the minimum threshold for detecting productively infected cells in lymph nodes by flow cytometry requires a plasma virus concentration of approximately 2.5 x 10(4) vRNA copy Equivalents (Eq)/ml. Conversely, an approximately 2 logs higher plasma viral load is needed to detect productively infected cells in the peripheral blood. This novel protocol provides a direct analytical tool to assess interactions between SIV and host cells, which is of key importance to investigators in AIDS research. (c) 2010 International Society for Advancement of Cytometry.

Anal Chem. 2010 Aug 15; 82(16): 6745-50
Xu X, Arriaga EA

Triphenylphosphonium hydroethidine (TPP-HE) is a membrane-permeable probe that reacts with superoxide and forms hydroxytriphenylphosphonium ethidium (OH-TPP-E(+)), a fluorescent product that has been previously used in qualitative measurements of superoxide production. In order to develop quantitative methods to measure superoxide, it is necessary to take into consideration the principles that drive TPP-HE accumulation into various subcellular compartments. In the mitochondria matrix, TPP-HE accumulation depends on the mitochondrial membrane potential, which varies from cell to cell. Here we address this issue by including rhodamine 123 (R123) as an internal mitochondrial membrane potential calibrant in chemical cytometry experiments. After loading with TPP-HE and R123, a single cell is lysed within a separation capillary and its contents are separated and detected by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). Using theoretical arguments, we show that the ratio [OH-TPP-E(+)]/[R123] is adequate to obtain a relative quantitation of mitochondrial matrix superoxide levels for each analyzed cell. We applied this method to single skeletal muscle myoblasts and determined that the steady state superoxide levels in the mitochondrial matrix is approximately (0.29 +/- 0.10) x 10(-12) M. The development of this quantitative method is a critical step toward establishing the importance of reactive oxygen species in biological systems, including those relevant to aging and disease.

J Phys Chem B. 2010 Aug 12; 114(31): 10010-6
Hema Sagar G, Tiwari MD, Bellare JR

Vesicles are usually characterized for their structure by microscopy or, less often, by the addition of fluorescent dyes in a flow cytometer. We present a new method of studying these structures and associated forms by forward and side scatter analysis on a flow cytometer which has the advantage of simultaneous handling of large population of vesicles to identify their shapes and lamellarities. The technique is suitable for several types of vesicular structures like Multivesicular vesicles (MVV), multilamellar and unilamellar vesicles. Characteristic signatures are given by tubular structures and fine features thereon allow detection of complex structures such as fused and ellipsoidal forms. Coexistence of tubular and spherical structures, such as those known to form when surfactants/salt solutions are diluted, can clearly be detected by the signature pattern, which separates into two distinctly identifiable populations. The population can be sorted or separated easily based on these signatures and such sorting has allowed us to confirm our findings by microscopic observations. This novel method can thus be used for concurrent observations of vesicle populations with dye or more advantageously without employing any fluorescent tag.

Cytometry A. 2010 Sep; 77(9): 861-72
Houston JP, Naivar MA, Freyer JP

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 mus, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees . The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 mus and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better exploitation of the traditionally underutilized parameter of fluorescence lifetime. Published 2010 Wiley-Liss, Inc.