Detection of cells by flow cytometry:Counting,imaging,and cell classification

The study of circulating cells in the blood stream is critical,as it covers many felds of biomed-icine,including immunology,cell biology,oncology,and reproductive medicine.In-viuo flowcytometry(IVFC)is a new tool to monitor and count cells in real time for long durations in theirnative biological environment.This review describes two main categories of IVFC,ie.,labeledand label-free IVFC.It focuses on label-free IVFC and introduces its technological developmentand related biological applications.Because cell recognition is the basis of flow cytometrycounting,this review also describes various methods for the classification of unlabeled cells,including the latest machine learning-based technologies.

Demands and technical developments of clinical flow cytometry with emphasis in quantitative,spectral,and imaging capabilities

As the gold-standard method for single-cell analysis,flow cytometry enables high-throughput and multiple-parameter characterization of individual biological cells.This review highlights the demands for clinical flow cytometry in laboratory hematology(e.g.,diagnoses of minimal residual disease and various types of leukemia),summarizes state-of-the-art clinical flow cytometers(e.g.,FACSLyricTMby Becton Dickinson,DxFLEX by Beckman Coulter),then considers innovative technical improvements in flow cytometry(including quantitative,spectral,and imaging approaches)to address the limitations of clinical flow cytometry in hematology diagnosis.Finally,driven by these clinical demands,future developments in clinical flow cytometry are suggested.

Performance Evaluation of Combined Detection of Serum CEA,CYFRA21-1,CA125,and NSE in Patients with Lung Cancer by Fluorescence Flow Cytometry

Objective:To investigate the effect of combined detection of serum carcinoembryonic antigen(CEA),cytokeratin 19 fragment(CYFRA21-1),cancer antigen 125(CA125),and neuron-specific enolase(NSE)in patients with lung cancer by fluorescence flow cytometry.Methods:From August 2019 to July 2022,200 patients with lung cancer diagnosed by pathology in our hospital were retrospectively analyzed.2 mL venous blood was collected in a fasting state and centrifuged to separate the serum(containing human chorionic gonadotropin antibody[anti-hCG antibody],hepatitis B surface antibody[anti-HBs antibody],and CEA).Results:The sensitivities of CEA and CYFRA21-1 detected via enzyme-linked immunosorbent assay(ELISA)were 100%,and the detection limits were 0.5 ng/mL and 0.1 ng/mL,respectively;the sensitivities of CA125 and NSE detected via flow cytometry were 100%,and the detection limits were 10 U/mL and 2 ng/mL,respectively.Compared with ELISA,the sensitivities of CA125 and NSE detected via flow cytometry were higher.When the concentration of CEA was 10-40 ng/mL,the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 40-80 ng/mL,the sensitivity of CEA significantly decreased(P<0.01),but the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 80-200 ng/mL,the sensitivities of all four markers showed no significant changes(P>0.05).Conclusion:Compared with the double-antibody sandwich ELISA,fluorescence flow cytometry has certain advantages,including high sensitivity,good precision,short detection time,low sample usage,and low medical cost;thus,it is worthy of clinical promotion.

The Clinical Value of Detecting the Level of Exfoliated Cells in Pleural Effusion by Flow Cytometry in the Differential Diagnosis of Non-

Objective:To explore the value of flow cytometry(FCM)in detecting the level of exfoliated cells in pleural effusion in the differential diagnosis of non-small cell lung cancer and benign lung diseases.Methods:Clinical data of patients with non-small cell lung cancer who were hospitalized in Hebei hospital from June 2019 to March 2022 were collected.A total of 98 patients were included,and 63 patients with alveolar lung disease were screened during the same period,and the two groups of patients were analyzed.Results:Compared with alveolar lung disease group,FCM detection and analysis showed that the level of exfoliated cells in the pleural effusion of non-small cell lung cancer(NSCLC)patients was 99(3-969)/100,000,and patients with alveolar lung disease was 4(0~19)/100,000.Additionally,compared with the alveolar lung disease group,the level of exfoliated cells in the pleural effusion of patients with non-small cell lung cancer(NSCLC)was significantly increased(P<0.001).The diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in non-small cell lung cancer was assessed using ROC curves and using 95%CI(-11.1,-13.2)with a sensitivity of 0.75 and specificity of 0.94,and the diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in alveolar lung disease was assessed using 95%CI(-11.1,-13.2)with a sensitivity of 0.71 and specificity of 0.87.Conclusion:Flow cytometry has a wider range of clinical applications,simple operation,low cost,and high sensitivity,which makes it of great significance in disease diagnosis.

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Recent advances in fuorescence-based in vivo flow cytometry

The fuorescence-based in vivo flow cytometry(IVFC)is an emerging tool to monitor eirculating cells in vivo.As a noninvasive and real-timne diagnostic technology,the fluorescence based IVFC allows long-term monitoring of circulating cells without changing their native biological environment.It has been applied for various biological applications(eg,monitoring circulating tumor cells).In this work,we will review our recent works on fluorescence-based IVFC.The operation principle and typical biological applications will be introduced.In addition,the recent advances in IVFC flow cytometry based on photoacoustic effects and other label free detection methods such as imaging based methods,difuse-light methods,hybrid multimodality methods and multispectral methods are also summarized.

Advances in small animal mesentery models for in vivo flow cytometry,dynamic microscopy,and drug screening

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Application of CD45/SSC Gating Multiparameter Flow Cytometry in the Classification of Acute Leukemia——An Analysis of 139 Cases

In order to study the significance of flow cytometry immunophenotyping in the diagnosis of acute leukemia, CD45/SSC gating multiparameter flow cytometry (FCM) was utilized to analyze the immunophenotypes of 139 cases of acute leukemia. 139 cases of acute leukemia were enrolled in our hospital from April 1998 to April 2000. Morphological analysis and FCM immunophenotypic tests were conducted on all cases. Our results showed that CD45/SSC gating multiparameter flow cytometry immunophenotyping could reflect the origin of leukemic cells specifically. It is one of the important methods for the diagnosis of ALL, AML, and HAL. CD45/SSC gating multiparameter FCM analysis is a good technique for immunophenotyping. FCM immunophenotypic analysis can help improve the diagnosis and classification of acute leukemia, and extend the use of FCM in clinical practice.

DETECTION OF PLATELET-DERIVED MICROPARTICLES USING FLOW CYTOMETRY AND ITS CLINICAL APPLICATION

Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extracted by gradual centrifugation. According to the size of PMPs,3 μm and 0.8μm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter. Results. In 30 healthy donors,the average concentration of resting PMPs was(1.2×105±5.7×104 )/ml and that of activated PMPs was(1.6×106±9.1×105)/ml. Compared with healthy donors,PMPs mean value was significantly higher(P< 0.001)in 18 patients with coronary artery disease,12 with acute cerebral infraction and 23 with chronic renal failure[the average PMPs concentration,( 6.1×105±2.5×105 )/ml, ( 6.8×105±3.4×105)/ml and(5.9×105±3.1×105)/ml respectively]. However,no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [1.3×105±6.1×104)/ml,(P >0.05)] .Conclusions. PMPs is a useful indicator in monitoring platelet activation,and plays an important role in thrombotic disease. By flow cytometric internal standard method,PMPs can be counted rapidly and accurately,which may be very helpful in interlaboratory comparative studies.

Flow cytometry for the assessment of animal sperm ntegrity and functionality： state of the art

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ＇bench-top＇ flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

Morphological Effects of Natural Products on Schizosaccharomyces pombe Measured by Imaging Flow Cytometry

Gaining a full understanding of the mechanisms of action of natural products as therapeutic agents includes observing the effects of natural products on cellular morphology,because abnormal cellular morphology is an important aspect of cellular transformations that occur as part of disease states.In this study a set of natural products was examined in search of small molecules that influence the cylindrical morphology of fission yeast Schizosaccharomyces pombe.Imaging flow cytometry of large populations of S.pombe exposed to natural products captured cell images and revealed changes in mean length and aspect ratio of cells.Several natural products were found to alter S.pombe’s morphology relative to control,in terms of elongating cells,shrinking them,or making them more round.These results may facilitate future investigations into methods by which cells establish and maintain specific shapes.

Characterization of ploidy levels in Chrysanthemum L. by flow cytometry

Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthe- mum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu moun- tain （Henan province）, and C. zawadskii was collected at Huangshan mountain （Anhui province）. We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to bea simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.

Analysis of DNA Content of Various Types of Spermatogenic Cells in Rat after Testicular Heating with Flow Cytometry

To measure DNA contents of spermatogenic cells and analyze the efficiency of spermatogenesis after testicular heating in rat Methods Eighty adult male Sprague-Dawley rats were randomly divided into experimental group （43 ℃, 30 min） and control group （22 ℃, 30 min）. According to day 0.5, 1, 3, 6, 10, 25, 35 and 50 after local testicular heating, every group was divided into 8 subgroups： experimental subgroups （n=6） and control subgroups （n=4）. DNA contents of various types of germ cells were observed with flow cytometry （FCM） in all groups. Results Compared with control groups, percentages of 4C cell （primary spermatocyte） in 0.5-35 d groups and percentages of 1C cell （spermatid and sperm） in 6-50 d groups significantly decreased in experimental groups （P〈0.05）, and percentages of 2C cell （spermatogonium and second spermatocyte） in 3 -35 d experimental groups increased significantly after heating （P〈0.05）. 4C：2C in all of 8 experimental groups and 1C：2C in 3-35 d experimental groups were down （P〈0. 05）, and in 1 d experimental group 1C：4C was up after heating （P〈0.05）. Conclusions After being heated, the number of spermatocyte firstly decreased, and then that of spermatid and sperm decreased too. Heat influences several stages in spermatogenesis and results in suppression of spermatogenesis. Flow cytometry is an effective method for researching on the change of spermatogenesis and has significance on mechanism about changing of spermatogenic cells induced by heat.

Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

Measurement of the Activation of IL-2 on Bone Marrow by Flow Cytometry

The changes of cell surface markers before and after activation by IL-2were detected by flow cytometry (FCM) to establish a more convenient and pre-cise criterion for the judgment of the activation of bone marrow. By using the measurement of the release of lactate dehydrogenase (LDH) the cytotoxicity of mononuclear cells (MNCs) from bone marrow, activated or inactivated, on tumor cell line K562 was evaluated, and at the same time the changes of surface markers on MNCs before and after activation were examined by using FCM. The results showed that the cytotoxicity of MNCs from bone marrow activated by IL-2 on tu-mor cell line K562 was increased obviously and the number of CD and CD posi-tive cells in bone marrow MNCs was higher than be fore activation. The enhanced cytotoxicity of MNCs on tumor cell line K562 was synchronous with the increaseof the number of CD and CD positive cells in 48 to 72 h. It is more direct, simple and precise to demonstrate the activation of IL-2 on bone marrow by detecting the changes of the amount of the CD and CD positive cells in bone marrow by FCM.

Diagnosis of Disorders of Platelet Function by Flow Cytometry

The platelet membrane glycoprotein (GP)Ⅱ b/Ⅲ a, GPIX were detected by immunogold assay and flow cytometry, respectively, in two cases of Glannzmann's thrombasthenia (GT). The immunogold assay showed that the GPⅡ b/Ⅲ a gold granules in GT were decreased obviously, compared with that in normal controls. Flow cytometry results showed that GPⅡ b/Ⅲ a in two cases of GT was 0.1 % and 0.5 % of normal control, respectively. While GPIX showed no difference between the GT and normal control. The patients' father showed no bleeding symptoms, whose GPⅡ b/Ⅲ a accounted for 36.99 % of normal control. The experiment results suggested that flow cytometry is a quick, simple and sensitive method for the diagnosis of GP disorders.

Use of flow cytometry to investigate the cytokine response pattern in infants with respiratory syncytial virus infection and bronchiolitis

Objective: To investigate the cytokine response pattern (IL 4/IFN γ ) in infants with RSV infections and bronchiolitis during the acute phase. Methods: Four color flow cytometry was used to measure intracellular IL 4 and IFN γ expressions in peripheral blood CD3+ and CD8+ lymphocytes from RSV infected and bronchiolitis infants. Serum IL 4 and IFN γ levels were also determined. Results: RSV infected and bronchiolitis infants showed no statistical differences from not RSV infected or pneumonia infants and control in the frequency of IL 4 and IFN γ expressions in CD3+CD8 lymphocytes, showed no obvious Th1/Th2 imbalance, while IFN γ was expressed much more frequently in CD3+CD8+ lymphocytes. Systematically, RSV infected and bronchiolitis infants showed much lower levels of serum IL 4 and IL 4/IFN γ ratios and much higher serum IFN γ levels than control. However, there were no statistical differences in the above three indices between RSV infected and not RSV infected infants or between bronchiolitis and pneumonia infants, except that bronchiolitis infants had a higher level of serum IFN γ than pneumonia infants statistically. Conclusions: There is no type 2 cytokine response predominance in the acute phase of RSV infection and bronchiolitis. IL 4 production is suppressed and IFN γ production upregulated, the latter being most prominent in bronchiolitis infants.

The feasibility of antitumor drugs chemosensitivity testing by flow cytometry

Objective: To investigate the feasibility of chemosensitivity testing of antitumor drugs by flow cytometry in clinical applications so as to provide experimental and theoretical basis for the establishment of a novel antitumor drugs sensitivity testing and the screening of particular antitumor drugs. Methods: Detect the apoptosis rate of 12 cases of Molt-4 cell line, 57 cases of fresh clinical gastrointestinal tumor cells by Sub-G1 and Annexin V assay of flow cytometry under the effects of antitumor drugs at different times and the outcomes were compared with the ones of the MTT (3-(4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) assay. Results: The lethality of drugs on Molt-4 cell, clinical gastrointestinal tumor cells had a positive correlation with the acting time of antidrugs by employing Annexin V, Sub-G1 and MTT assay. Drug-incurring maximum lethality of Annexin V assay was higher than MTT colorimetric assay, that of Sub-G1 was lower than MTT assay, the virtual times of Annexin V and Sub-G1 assay were obviously earlier than that of MTT colorimetric assay. Conclusion: Annexin V and Sub-G1 assay of flow cytometry can be taken as potent protocols testing anti-tumor drug chemosensitivity. Annexin V assay is featured by more sensitive, concise, reliable compared with the classical chemosensitivity testing assay of MTT colorimetric assay and it possesses clinical applied value.