A quantitative microassay for leukocyte chemotaxis,using a microscopic slide system with complement-activating yeast particles as gradient source

A simple quantitative microassay was developed for studying polymorphonuclear leukocyte (PMNL) chemotaxis under conditions where the number of available cells is a limiting factor, e.g., pustules, neutropenia, small children and cerebrospinal fluid.PMNL suspensions are placed on glass slides to which fluorescein-labeled yeast particles have been fixed. After adherence, normal human serum is added to the slides. Owing to complement activation, a chemotactic gradient which attracts the adherent PMNL is formed around the yeast particles. The number of PMNL-associated yeast particles in the presence of normal serum is scored, and compared with cells migrating in the presence of inactivated serum or in the absence of serum. A locomotory index is calculated as the number of yeast particles associated with PMNL divided by the total number of yeast particles.     

The effect of chemotaxis and chemokinesis on leukocyte locomotion: A new interpretation of experimental results

A mathematical model is developed to describe the motion ofleukocytes through a Boyden chamber. The model is based on theKeller–Segel model of cell motion and comprises threepartial differential equations which describe the evolutionof the neutrophils, the chemoat-tractant, and a neutrophil-derivedchemokinetic factor. Where other authors have concentrated onchemotaxis, here attention is focused on the manner in whichthe chemokinetic factor influences neutrophil locomotion. Numericalsimulations show how the number of neutrophils initially placedon top of the chamber affects cellular motion through the systemand reproduce the qualitative behaviour observed by Takeuchi& Persellin (Am. J. Physiol. 236, C22–C29). In particular,the simulations show how dense packing of the neutrophils increasesthe levels of the chemokinetic factor. This enhances randomcell motion and increases the speed with which the neutrophilsreach the source of chemoattractant. For a particular asymptoticlimit of the system parameters, the model reduces to a nonlinearpartial differential equation for the neutrophils. Similaritysolutions of this caricature model yield algebraic expressionsrelating the speed with which the neutrophil front penetratesinto the chamber to the number of neutrophils initially placedon top of it. The implications of the results are also discussed.     

Role of DNA flow cytometry and immunocytochemical analysis in diagnosis of malignant effusions

Body cavity fluid examination sometimes presents a diagnostic challenge in cytology practice. This study was undertaken to evaluate efficacy of cytomorphology, epithelial membrane antigen immunocytochemistry (EMA-ICC) and DNA flow cytometry (FCM) in detection of malignant cells in effusions. One hundred effusions (55 pleural, 44 ascitic, and 1 pericardial fluid) were studied by cytology, EMA, and FCM. There were 29 malignant and 71 benign cases. On cytology, 28 of 29 malignant cases were diagnosed. With no false positives, the sensitivity and specificity was 96.55% and 100% respectively. FCM detected aneuploidy in 85.71% of cytologically malignant and 4.17% of cytologically benign effusions. EMA was positive in 75% of cytologically malignant and 4.17% of cytologically benign cases. EMA had lower sensitivity than cytology; 75.86% versus 96.55%. Sensitivity and specificity of FCM was 86.21%, and 97.18% respectively. FCM had lower sensitivity than cytology; 86.21% versus 96.55%. Sensitivity increased to 100% (P < 0.05) when the combinations of cytology plus EMA or cytology plus ploidy were applied compared to cytology alone (96.55%). Also, the combination of cytology plus EMA had higher sensitivity than EMA alone (100% versus 75.86%, P < 0.05) and combined cytology plus ploidy had higher sensitivity than ploidy alone (100% versus 86.21%, P < 0.05). The study demonstrates the usefulness of EMA-ICC and DNA FCM as adjuncts to cytology to diagnose malignancy in effusions. FCM in combination with ICC can be further developed to reduce number of false-negative cases on cytology and add objectivity to cytologically doubtful or equivocal cases.     

Isolation and characterization of endometrial leukocyte populations by flow cytometry

Potentialities of the flow cytornetry method in studies of the qualitative and quantitative composition of endometrial immunocompetent cells are demonstrated. The studied leukocyte populations are found to occur in negligible quantities in the endometrium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 1, pp. 68–71, January, 1994.     

A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry

A large number of purified and washed PMNLs are required to monitor generation of active oxygen derivatives in most in vitro studies and this can preclude investigations in small children. The present method has enabled us to measure the oxidative burst (generation of hydrogen peroxide) of PMNLs in a small amount of whole blood using 2′,7′-dichlorofluorescin diacetate, phorbol myristate acetate and flow cytometry. Optimal conditions for this determination were evaluated and the reaction was found to be independent of the absolute numbers of PMNLs and other types of cell in whole blood. The present method will be of value in investigations of the leukocyte metabolism of patients not only with chronic granulomatous disease (CGD) but also with various infectious diseases.     

Detection of antisperm antibodies by immobilization and cytotoxicity tests using flow cytometry

All-Union Research Institute for Maternal and Child Health Care, Ministry of Health of Russia. Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 10, pp. 398–399, October, 1992.     

Lymphoid and myeloid neoplasms involving cerebrospinal fluid: comparison of morphologic examination and immunophenotyping by flow cytometry

We studied 53 samples of cerebrospinal fluid (CSF) by cytologic examination and immunophenotyping by flow cytometry. The samples were taken from 43 patients; 25 had a previous diagnosis of malignant lymphoma/leukemia and the remaining 18 a variety of other diseases involving the central nervous system (CNS). Lymphoma/leukemia was detected in 21 samples: 12 by morphologic examination and immunophenotyping and nine by immunophenotyping alone. There were two cases with a suspicious morphologic examination and negative immunophenotyping in which the final diagnosis were cryptococcal and viral meningitis. In the group of 18 patients, one was diagnosed as a primary malignant lymphoma of the CNS and was positive with cytology and immunophenotyping. The other 17 were negative with both methods and follow-up showed no evidence of lymphoma/leukemia. This study shows that morphologic examination combined with flow cytometry enhances the detection rate by 75% over morphologic examination alone in CSF samples.     

采用流式细胞术检测血小板微粒及其临床应用的初探

本研究应用流式细胞仪（FCM）,以3μm 和0.8μm 的标准微球作内参对照建立了血小板微粒（PMPs）的定量检测方法,并对其临床应用进行了初步研究。结果表明：30 例健康人静止血小板释放的PMPs 为1.2×10~5± 5.7×10~4 /mL,活化血小板释放的PMPs 为1.6×10~ 6±9.1×10~5 /mL,18 例冠心病CAD 12 例急性脑梗塞（ACI）和23 例慢性肾功衰竭（CRF）患者血浆中PMPs 较对照组明显增高（P<0.001）,分别为6.1×10~5±2.5×10~5 /mL、6.8×10~5± 3.4×10~5 /mL 和5.9×10~5± 3.1×10~5 /mL, 而25 例急性白血病（AL）化疗后伴严重血小板减少者的PMPs 增高不明显,为1.3×10~5±6.1 ×10~4/mL。提示：PMPs 是监测血小板活化、预测和诊断血栓性疾病的一个重要指标,采用FCM 内参定位法可以准确而快速地定量分析PMPs,有利于实验结果的室间比较和检测方法的标准化。     

Clinical role of flow cytometry in redefining bone marrow involvement in diffuse large B-cell lymphoma (DLBCL) - a new perspective

Aims:  The clinical role of flow cytometry in staging bone marrow in diffuse large B-cell lymphoma (DLBCL), especially its impact on outcome, remains uncertain. The aim was to determine the contribution of flow cytometry to conventional staging, and to study the impact of this revised staging on survival.
Methods and results:  One hundred and thirteen cases of DLBCL diagnosed at The Canberra Hospital from 1996 to 2005 were identified. Blinded analysis of bone marrow (BM) morphology and flow cytometric data showed involvement on morphology (M) in 25 (22.1%) cases, on flow cytometry (F) in 21 (18.6%) cases and overall (M + F) in 32 cases (28.3%); discordance was noted in 16 cases (16.1%). Cases with and without marrow involvement on conventional staging alone (M) had no significant difference in survival ( P  = NS). However, when BM involvement was defined as positivity on morphology and/or flow cytometry (M + F), the median survival of patients with involvement was significantly worse than patients without involvement ( P  = 0.026).
Conclusions:  Flow cytometry-positive cases should be included with those positive on morphology in a summative model to define BM involvement in DLBCL, as it may have a potential impact on predicting outcome.     

A flow cytometry based in vitro micronucleus assay in TK6 cells—Validation using early stage pharmaceutical development compounds

The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assay's advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss,Inc.     

Diagnosis of posttransplant granulocytic sarcoma by fine‐needle aspiration cytology and flow cytometry

We describe a patient who developed granulocytic sarcomas of the mesentery and breast approximately 4 yrs following an allogenic bone marrow transplantation for acute myeloblastic leukemia. The diagnosis was made by a combination of fine‐needle aspiration cytology and flow cytometry. The differential diagnoses of localized masses in posttransplant patients and how the combination of fine‐needle aspiration cytology and flow cytometry may be used are discussed. Diagn Cytopathol 1999;20:85–89. © 1999 Wiley‐Liss, Inc.     

A quantitative exploration of surface antigen expression in common B-cell malignancies using flow cytometry

The use of flow cytometry to diagnose hematological malignancies has become routine due to its ability to often differentiate between morphologically similar diseases based on antigens expressed on the surface of malignant cells. In an attempt to expand on the utility of flow cytometry in the study of B-cell malignancies we have used the most reliable quantitative methodology, QIFI (quantitative indirect immunoflourescence assay), to study the expression of CD5, CD10, CD11c, CD19, CD20, CD22, CD23, and CD79b in 384 cases of several common B-lineage malignancies, including: B-ALL, CLL, SLL, hairy cell leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. The impetus behind this extensive, single institution study of surface antigens was two-fold: evaluating similarities and differences of antigen expression between B-cell neoplasms and finding additional clinical utility for the quantitative flow cytometric data generated. Our results show that each distinct malignant histology has its own quantitative pattern of surface antigen expression. In most cases, these quantitative patterns do not increase the ability of flow cytometry to distinguish between them. However, a high expression of specific antigens on a given B-cell malignancy may potentially identify optimal therapeutic targets for current and/or future monoclonal antibody-based therapies.     

Diagnosis of myeloid sarcoma involving salivary glands by fine-needle aspiration cytology and flow cytometry: report of four cases

Myeloid sarcoma involving salivary glands is extremely rare. Here, we report four cases of this rare occurrence, diagnosed by fine-needle aspiration biopsy. All of four patients had previous diagnoses of myeloid neoplasms. They presented with a solitary mass in the parotid or submandibular salivary gland. The cytological evaluation of the aspirates revealed scattered salivary gland acini admixed with dispersed atypical cells. In three cases, the atypical cells appeared to be heterogeneous, intermediate to large in size, and have folded nuclei with fine chromatin. In another case the atypical cells were monotonous and had round nuclei with fine chromatin. The myeloid lineage of the atypical cells was demonstrated by flow cytometric analysis. High clinical suspicion, careful cytological evaluation, and concurrent ancillary studies are essential for establishing a diagnosis of myeloid sarcoma.     

A comparative study of proliferation indices and ploidy in dysplastic naevi and malignant melanomas using flow cytometry

Cell proliferation indices and DNA content have been determined in 18 intradermal naevi, 40 dysplastic naevi and 16 superficial malignant melanomas (less than 0.76 mm depth of invasion) using flow cytometry. In this study, proliferation indices of intradermal naevi and dysplastic naevi were not significantly different from each other. Abnormalities of DNA ploidy were not identified in the intradermal naevi or dysplastic naevi; whereas three of the malignant melanomas were aneuploid. In addition, cellular proliferation was increased within the group of malignant melanomas, in comparison with the naevi. This study has found no evidence to indicate that sporadic dysplastic naevi were more likely than intradermal naevi to transform to malignant melanoma, when objective criteria were employed. However, dysplastic naevi could be distinguished from some early malignant melanomas by absence of aneuploidy and by low cell proliferation indices.     

A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of bla_TEM, bla_SHV or bla_CTX-M genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.     

A rapid method for infectivity titration of Andes hantavirus using flow cytometry

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24–48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples.     

A chromatic explosion: the development and future of multiparameter flow cytometry

Multiparameter flow cytometry has matured tremendously since the 1990s, giving rise to a technology that allows us to study the immune system in unprecedented detail. In this article, we review the development of hardware, reagents, and data analysis tools for multiparameter flow cytometry and discuss future advances in the field. Finally, we highlight new applications that use this technology to reveal previously unappreciated aspects of cell biology and immunity.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.